Mediator Of Endoplasmic Reticulum Stress Research Articles

Chang'an decoction alleviates endoplasmic reticulum stress by regulating mitofusin 2 to improve colitis.

To evaluate the protective effects of Chang'an decoction (, CAD) on colitis, and investigate the potential mechanisms underlying these effects from the perspectives of endoplasmic reticulum (ER) stress induced by mitofusin 2 (MFN2). The composition of CAD was identified by liquid chromatography-mass spectrometry technology. A mice model of dextran sulfate sodium (DSS) induced colitis was established and therapeutic effects of CAD were determined by detecting body weight, disease activity index, colon length and histopathological changes. Then, the expression levels of MFN2, ER stress markers and Nucleotide-binding domain and leucine-rich repeat protein3 (NLRP3) relevant proteins were detected by polymerase chain reaction (PCR), Western blot, immunohistochemistry and immunofluorescence staining. Subsequently, knockdown and overexpression cell model were constructed to further investigate the underlying mechanism of MFN2 mediating ER stress and energy metabolism by PCR, Western blot, electron microscopy and reactive oxygen species (ROS) staining. Finally, inflammatory indicator and tight junction proteins were measured by PCR and immunofluorescence staining to evaluate the protective effects of CAD. Results showed that the indispensable regulatory role of MFN2 in mediating ER stress and mitochondrial damage was involved in the protective effects of CAD on colitis in mice fed with DSS. Network pharmacology analysis also revealed CAD may play a protective effect on colitis by affecting mitochondrial function. In addition, our data also suggested a causative role for MFN2 in the development of inflammatory responses and energy metabolic alterations by constructing a knockdown and overexpression cell model whereby alter proper ER-mitochondria interaction in Caco-2 cells. Furthermore, relative expression analyses of ER stress markers and NLRP3 inflammasome showed the onset of ER stress and activation of NLRP3 inflammasome, which is consistent with the above findings. In contrast, intervention of CAD could improve the mucosal barrier integrity and colonic inflammatory response effectively through inhibiting ER stress response mediated by MFN2. CAD could alleviate ER stress by regulating MFN2 to exert therapeutic effects on DSS-induced colitis, which might provide an effective natural therapeutic approach for the treatment of ulcerative colitis.

Hhatl ameliorates endoplasmic reticulum stress through autophagy by associating with LC3

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.

Molecular mechanism of apoptosis induced by 4-tBP in common carp (Cyprinus carpio L.) head kidneys was explored from various angles: Hippo pathway, miR-203a, oxidative stress, ER stress, and mitochondrial pathway

4-tert butylphenol (4-tBP), a toxic environmental pollutant estrogen, received widespread attention due to its widespread presence, environmental persistence, and toxic effect. Common carp (Cyprinus carpio L.) is the most important freshwater economic animal in China and is also a model animal for the study on molecular mechanisms of poisoning. Head kidney is a target organ attacked by environmental pollutants. Here, we established a common carp 4-tBP poisoning model. The results showed that exposure to 4-tBP resulted in mitochondrial damage, endoplasmic reticulum damage, and apoptotic damage in head kidneys of common carps using microstructure and ultrastructure observations, as well as TUNEL kit methods. Furthermore, we found at transcriptional level from three perspectives that 4-tBP induced apoptosis in common carp head kidney cells: Hippo pathway, mitochondrial pathway, and ER stress. Hippo pathway formed a complex network, in which LATS1 was a core gene. It's noteworthy that Hippo pathway triggered mitochondrial pathway through YAP/NR4A1/Bcl-2 and YAP/BIRC5/Caspase-9 pathways under 4-tBP-induced apoptosis. PERK-eIF2α-ATF4-Caspase-3 pathway was involved in 4-tBP-induced apoptosis via ER stress. Moreover, miR-203a mediated 4-tBP-induced apoptosis through targeting MST1, MAP4K4, and NR4A1. In addition, 4-tBP disrupted balance between oxidants and antioxidants, and caused oxidative stress, and oxidative stress mediated ER stress under 4-tBP-caused apoptosis via via H2O2-PERK-eIF2α-ATF4-Caspase-3 pathway. miR-203a and oxidative stress participated in 4-tBP-induced apoptosis in common carp head kidney cells. For the first time, we found that Hippo pathway took part in molecular mechanism of fish poisoning, which needs to be investigated in the future. Our research provided foundational knowledge for exploring toxic effects of estrogen on fish and provided new insights into a theoretical basis for safety risk assessment for humans and fish, which deserves further profound exploration in the future. Moreover, the insights we generated help inform new strategies for preventing toxic injury in fish.

Cholestane-3β,5α,6β-triol Induces Multiple Cell Death in A549 Cells via ER Stress and Autophagy Activation.

Cholestane-3β,5α,6β-triol (CT) and its analogues are abundant in natural sources and are reported to demonstrate cytotoxicity toward different kinds of tumor cells without a deep probe into their mechanism of action. CT is also one of the major metabolic oxysterols of cholesterol in mammals and is found to accumulate in various diseases. An extensive exploration of the biological roles of CT over the past few decades has established its identity as an apoptosis inducer. In this study, the effects of CT on A549 cell death were investigated through cell viability assays. RNA-sequencing analysis and western blot of CT-treated A549 cells revealed the role of CT in inducing endoplasmic reticulum (ER) stress response and enhancing autophagy flux, suggesting a putative mechanism of CT-induced cell-death activation involving reactive oxygen species (ROS)-mediated ER stress and autophagy. It is reported for the first time that the upregulation of autophagy induced by CT can serve as a cellular cytotoxicity response in accelerating CT-induced cell death in A549 cells. This research provides evidence for the effect of CT as an oxysterol in cell response to oxidative damage and allows for a deep understanding of cholesterol in its response in an oxidative stress environment that commonly occurs in the progression of various diseases.

Morroniside Protects C2C12 Myoblasts from Oxidative Damage Caused by ROS-Mediated Mitochondrial Damage and Induction of Endoplasmic Reticulum Stress.

Oxidative stress contributes to the onset of chronic diseases in various organs, including muscles. Morroniside, a type of iridoid glycoside contained in Cornus officinalis, is reported to have advantages as a natural compound that prevents various diseases. However, the question of whether this phytochemical exerts any inhibitory effect against oxidative stress in muscle cells has not been well reported. Therefore, the current study aimed to evaluate whether morroniside can protect against oxidative damage induced by hydrogen peroxide (H2O2) in murine C2C12 myoblasts. Our results demonstrate that morroniside pretreatment was able to inhibit cytotoxicity while suppressing H2O2-induced DNA damage and apoptosis. Morroniside also significantly improved the antioxidant capacity in H2O2-challenged C2C12 cells by blocking the production of cellular reactive oxygen species and mitochondrial superoxide and increasing glutathione production. In addition, H2O2-induced mitochondrial damage and endoplasmic reticulum (ER) stress were effectively attenuated by morroniside pretreatment, inhibiting cytoplasmic leakage of cytochrome c and expression of ER stress-related proteins. Furthermore, morroniside neutralized H2O2-mediated calcium (Ca2+) overload in mitochondria and mitigated the expression of calpains, cytosolic Ca2+-dependent proteases. Collectively, these findings demonstrate that morroniside protected against mitochondrial impairment and Ca2+-mediated ER stress by minimizing oxidative stress, thereby inhibiting H2O2-induced cytotoxicity in C2C12 myoblasts.

Post-treatment with maropitant reduces oxidative stress, endoplasmic reticulum stress and neuroinflammation on peripheral nerve injury in rats.

To determine the effective dose and therapeutic potential of maropitant using through expression of mediators of oxidative stress, inflammatory and of the unfolded protein response (UPR) (bio) markers on spinal cord using a model of neuropathic pain induced through chronic constriction injury (CCI) in rats. Randomized, blinded, prospective experimental study. 98 male Wistar rats. Rats were anesthetized with sevoflurane and after CCI, they were randomly assigned to the following groups that received: vehicle, 3, 6, 15, 30 e 50 mg/kg/24q of maropitant. The effect on inflammatory mediators (IL10, TNFα), oxidative stress (GPx, CAT, SOD), microglial (IBA-1) and neuronal (NeuN, TACR1) markers was evaluated though immunohistochemistry and expression levels of markers of hypoxia (HIF1α, Nrf2), antioxidant enzymes (Catalse, Sod1 and GPx1), and endoplasmic reticulum stress mediators (GRP78, CHOP and PERK) through qRT-PCR. Intraperitoneal injection (IP) of maropitant inhibited nociception with ID50 values of 4,1 mg/kg (5,85-19,36) in a neuropathic pain model through CCI. A dose of 30 mg/kg/24q was significantly effective in reducing mechanical allodynia 1 to 4h after treatment with nociception inhibition (145,83%). A reduction in the expression of hypoxia factors (HIF1α, Nrf2) was observed, along with an increase in antioxidant activity (CAT, SOD and GPX). Additionally, there was a reduction in inflammatory markes (IL10, TNFα), microglial (IBA-1), and neuronal markers (NeuN, TACR1). These findings demonstrate that the determined dose, administered daily for seven days, had an antinociceptive effect, as well as anti-inflammatory and antioxidant activity.

A new insight into Cd exposure-induced hemocyte reduction in Lymantria dispar larvae: Involvement of the ROS-ATF6-ER stress-apoptosis pathway

Hemocytes are important targets for heavy metal-induced immunotoxicity in insects. This study aimed to investigate the mechanism by which cadmium (Cd) exposure affects the hemocyte count in Lymantria dispar larvae. The results showed that the number of larval hemocytes was significantly decreased under Cd exposure, accompanied by a significant increase in the apoptosis rate and the expression of Caspase-3. The endoplasmic reticulum (ER) of hemocytes in the Cd-treated group showed irregular swelling. Expression levels of ER stress indicator genes (CHOP, Bip1, Bip2, Bip3, and Bip4) were significantly higher in the Cd-treated group. Among the three pathways that potentially mediate ER stress, only the key genes in the ATF6 pathway (ATF6, S1P-1, S1P-2, and WFS1) exhibited differential responses to Cd exposure. Cd exposure significantly increased the levels of reactive oxygen species (ROS) and the expression of oxidative stress-related genes (CNCC, P38, and ATF2) in hemocytes. Studies using inhibitors confirmed that apoptosis mediated the decrease in hemocyte count, ER stress mediated apoptosis, ATF6 pathway mediated ER stress, and ROS or oxidative stress mediated ER stress through the activation of the ATF6 pathway. Taken together, the ROS-ATF6-ER stress-apoptosis pathway is responsible for the reduction in the hemocyte count of Cd-treated L. dispar larvae.

MHY446 induces apoptosis via reactive oxygen species-mediated endoplasmic reticulum stress in HCT116 human colorectal cancer cells

This study investigated the potential of a newly synthesized histone deacetylase (HDAC) inhibitor, MHY446, in inducing cell death in HCT116 colorectal cancer cells and compared its activity with that of suberoylanilide hydroxamic acid (SAHA), a well-known HDAC inhibitor. The results showed that MHY446 increased the acetylation of histones H3 and H4 and decreased the expression and activity of HDAC proteins in HCT116 cells. Additionally, MHY446 was confirmed to bind more strongly to HDAC1 than HDAC2 and inhibit its activity. In vivo experiments using nude mice revealed that MHY446 was as effective as SAHA in inhibiting HCT116 cell-grafted tumor growth. This study also evaluated the biological effects of MHY446 on cell survival and death pathways. The reactive oxygen species (ROS) scavenger N-acetyl-L-cysteine (NAC) confirmed that ROS play a role in MHY446-induced cell death by reducing poly(ADP-ribose) polymerase cleavage. MHY446 also induced cell death via endoplasmic reticulum (ER) stress by increasing the expression of ER stress-related proteins. NAC treatment decreased the expression of ER stress-related proteins, indicating that ROS mediate ER stress as an upstream signaling pathway and induce cell death. While MHY446 did not exhibit superior HDAC inhibition efficacy compared to SAHA, it is anticipated to provide innovative insights into the future development of therapeutic agents for human CRC by offering novel chemical structure-activity relationship-related information.

PSMC2 Overexpression Enhances Proteasome Activity, Mitigates ER Stress, and Correleates with Reduced Overall Survival in Multiple Myeloma

Background: Multiple myeloma (MM) remains a highly treatable but generally incurable plasma cell dyscrasia. These plasma cells synthesize massive amounts of paraprotein relying heavily on the ubiquitin proteasome system to maintain homeostasis and are exquisitely sensitive to proteasome inhibition to avoid terminal ER stress. We postulated that increased expression of clinically relevant proteasome subunits would correlate with reduced overall survival in MM patients treated with proteasome inhibitors. Objective: To correlate proteasome gene expression levels to clinicals outcomes in both the phase III APEX trial and the MMRF COMMPASS Trial. To model the effects of these hits on MMCL UPS function. Results: We identified that 19S regulatory ATPases PSMC2 and PSMC6 correlated with poorer OS, outperforming the proteasome inhibitor (PI) target, PSMB5, in this analysis. We focused on modeling the more frequently upregulated PSMC2. We created PSMC2 overexpressing multiple myeloma cell lines, noting PSMC2 is readily incorporated in the higher order proteasome complexes. PSMC2 overexpressing cells exhibit higher levels of 20S proteasome activity (1.6 fold increase in ARH77 and 3 fold increase in U266 cell line compared to control, p< 0.01 in each case) and are more resistant to PI challenge (Bortezomib LD 50 of 30 nM v 15 nM in U266 overexpressed and control cells respectively, Bortezomib LD 50 of 18 nM v 10 nM in ARH77 overexpressed v control cells respectively). PIs are known to increase ER stress, and PSMC2 overexpressing cells are better able to mitigate PI mediated ER stress with PSMC2 overexpressing cells exhibiting less induction of ER stress sensors p-IRE1α and ATF4 compared to PSMC2 knockdown cells on bortezomib or tunicamycin challenge as quantitated by western blotting and utilizing an fluorescent ER stress sensor that models the conversion of XBP1u to XBP1s by pIRE1α. Mechanistically, PSMC subunits play a key role in the recognition and degradation of misfolded ER proteins, and increased 19S subunits may allow more effective ER associated protein degradation (ERAD) Conclusions: Taken together, our results suggest PSMC2 overexpression correlates with reduced OS in MM patients treated with bortezomib and that PSMC2 may represent an actionable therapeutic target to prevent or overcome PI resistance. PSMC2 overexpression increases proteasome activity in MM cells, mitigates ER stress on PI challenge and promotes PI resistance. mitigates ER stress upon PI challenge, and portending poorer clinical prognosis and more drug resistance, suggesting that 19S targeted agents may help overcome drug resistance in MM. Figure 1 A. PSMC2 overexpression increases chymotrypsin-like proteasome activity at baseline in U266 and ARH77 cells with a similar effect of residual proteasome activity after challenge with bortezomib or carfilzomib. B. ARH77 and U266 FLAG and vector cells were challenged with bortezomib for 18 hrs and then assayed by annexin PI staining. LD50 increased from 10 nm to 15 nm in ARH77 cells and from 12 to 25 in U266 Cells.

The amphiregulin/EGFR axis has limited contribution in controlling autoimmune diabetes

Conventional immunosuppressive functions of CD4+Foxp3+ regulatory T cells (Tregs) in type 1 diabetes (T1D) pathogenesis have been well described, but whether Tregs have additional non-immunological functions supporting tissue homeostasis in pancreatic islets is unknown. Within the last decade novel tissue repair functions have been ascribed to Tregs. One function is production of the epidermal growth factor receptor (EGFR) ligand, amphiregulin, which promotes tissue repair in response to inflammatory or mechanical tissue injury. However, whether such pathways are engaged during autoimmune diabetes and promote tissue repair is undetermined. Previously, we observed that upregulation of amphiregulin at the transcriptional level was associated with functional Treg populations in the non-obese diabetic (NOD) mouse model of T1D. From this we postulated that amphiregulin promoted islet tissue repair and slowed the progression of diabetes in NOD mice. Here, we report that islet-infiltrating Tregs have increased capacity to produce amphiregulin, and that both Tregs and beta cells express EGFR. Moreover, we show that amphiregulin can directly modulate mediators of endoplasmic reticulum stress in beta cells. Despite this, NOD amphiregulin deficient mice showed no acceleration of spontaneous autoimmune diabetes. Taken together, the data suggest that the ability for amphiregulin to affect the progression of autoimmune diabetes is limited.

Long noncoding RNA TRABA suppresses β-glucosidase-encoding BGLU24 to promote salt tolerance in cotton.

Salt stress severely damages the growth and yield of crops. Recently, long noncoding RNAs (lncRNAs) were demonstrated to regulate various biological processes and responses to environmental stresses. However, the regulatory mechanisms of lncRNAs in cotton (Gossypium hirsutum) response to salt stress are still poorly understood. Here, we observed that a lncRNA, trans acting of BGLU24 by lncRNA (TRABA), was highly expressed while GhBGLU24-A was weakly expressed in a salt-tolerant cotton accession (DM37) compared to a salt-sensitive accession (TM-1). Using TRABA as an effector and proGhBGLU24-A-driven GUS as a reporter, we showed that TRABA suppressed GhBGLU24-A promoter activity in double transgenic Arabidopsis (Arabidopsis thaliana), which explained why GhBGLU24-A was weakly expressed in the salt-tolerant accession compared to the salt-sensitive accession. GhBGLU24-A encodes an endoplasmic reticulum (ER)-localized β-glucosidase that responds to salt stress. Further investigation revealed that GhBGLU24-A interacted with RING-type E3 ubiquitin ligase (GhRUBL). Virus-induced gene silencing (VIGS) and transgenic Arabidopsis studies revealed that both GhBGLU24-A and GhRUBL diminish plant tolerance to salt stress and ER stress. Based on its substantial effect on ER-related degradation (ERAD)-associated gene expression, GhBGLU24-A mediates ER stress likely through the ERAD pathway. These findings provide insights into the regulatory role of the lncRNA TRABA in modulating salt and ER stresses in cotton and have potential implications for developing more resilient crops.

Abstract P3083: Trim29 Deficiency Ameliorates Viral Myocarditis By Reducing Perk Mediated Er Stress And Ros Responses

Infections by viruses including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) could cause viral myocarditis. Endoplasmic reticulum (ER) stress is associated with several metabolic diseases such as cardiovascular disease, diabetes and cancer. However, the immune mechanisms of ER stress regulation in controlling the pathogenesis of viral myocarditis are unclear. Here, we found that knockdown or knockout of TRIM29 significantly inhibited PERK (Protein kinase RNA-like endoplasmic reticulum kinase) mediated ER stress and apoptosis induced by cardiotropic viruses CVB3 (coxsackievirus B3) or EMCV (encephalomyocarditis virus) in human and mouse cardiomyocytes. Additionally, TRIM29 deficiency relieved ROS (Reactive oxygen species) mediated STING (Stimulator of interferon genes) inhibition to produce more type I interferon for restricting cardiotropic viruses in cardiomyocytes. Importantly, cardiomyocytes specific TRIM29 deficiency exhibited dramatic protective effects on cardiac damage, cardiac dysfunction, viral replication and promoted survival in CVB3-induced viral myocarditis model in vivo . Mechanistically, TRIM29 was shown to interact with PERK and catalyze SUMOylation for maintaining its stability, thereby promoting PERK mediated ER stress and ROS responses to induce pathogenesis of viral myocarditis. Our results indicate that TRIM29 loss-of-function in cardiomyocytes ameliorates viral myocarditis by reducing PERK mediated ER stress response and unleashing ROS-suppressed interferon response. Our data suggest the TRIM29-PERK axis is a potential therapeutic target for viral myocarditis and other ER stress-associated cardiovascular disease.

AMPK activation improves recovery from pneumonia-induced lung injury via reduction of er-stress and apoptosis in alveolar epithelial cells

BackgroundBacterial pneumonia and related lung injury are among the most frequent causes of mortality in intensive care units, but also inflict serious and prolonged respiratory complications among survivors. Given that endoplasmic reticulum (ER) stress is a hallmark of sepsis-related alveolar epithelial cell (AEC) dysfunction, we tested if AMP-activated protein kinase (AMPK) affects recovery from ER stress and apoptosis of AECs during post-bacterial infection.MethodsIn a murine model of lung injury by P. aeruginosa non-lethal infection, therapeutic interventions included AMPK activator metformin or GSK-3β inhibitor Tideglusib for 96 h. Recovery from AEC injury was evidenced by accumulation of soluble T-1α (AEC Type 1 marker) in BAL fluids along with fluorescence analysis of ER-stress (CHOP) and apoptosis (TUNEL) in lung sections. AMPK phosphorylation status and mediators of ER stress were determined via Immunoblot analysis from lung homogenates. Macrophage-dependent clearance of apoptotic cells was determined using flow cytometry assay.ResultsP. aeruginosa-induced lung injury resulted in accumulation of neutrophils and cellular debris in the alveolar space along with persistent (96 h) ER-stress and apoptosis of AECs. While lung infection triggered AMPK inactivation (de-phosphorylation of Thr172-AMPK), metformin and Tideglusib promptly restored the AMPK activation status. In post infected mice, AMPK activation reduced indices of lung injury, ER stress and related apoptosis of AECs, as early as 24 h post administration of AMPK activators. In addition, we demonstrate that the extent of apoptotic cell accumulation is also dependent on AMPK-mediated clearance of apoptotic cells by macrophages.ConclusionsOur study provides important insights into AMPK function in the preservation of AEC viability after bacterial infection, in particular due reduction of ER-stress and apoptosis, thereby promoting effective recovery from lung injury after pneumonia.

Scalp acupuncture alleviates cerebral ischemic stroke-induced motor dysfunction in rats via regulating endoplasmic reticulum stress and ER-phagy

Cerebral ischemic stroke is a high-risk disease and imposes heavy burdens on patients in china. Acupuncture has been used for thousands of years to treat motor dysfunction, cognitive disorder and language barrier caused by cerebral ischemic stroke. Acupoint lines, vertex middle line and anterior oblique line of vertex temple, are always employed to treat cerebral ischemic stroke. However, the mechanism of the two acupoint lines in relieving cerebral ischemic stroke needs further exploration. In the present study, scalp acupuncture treatment alleviated the motor dysfunction, brain damage, and cell death induced by middle cerebral artery occlusion (MCAO) in rats. Proteomics analysis and ultrastructure observation indicated that endoplasmic reticulum and lysosomes might involve in the mechanism of the scalp acupuncture treatment in suppressing MCAO-triggered neural deficits. Effect of the scalp acupuncture treatment on ER stress was then investigated and found that the activation of ER stress mediators, including PERK, IRE1, and ATF6, was downregulated after the scalp acupuncture treatment. Co-localisation analysis of KDEL and CD63 showed that the engulfment of ER fragments by lysosomes was accelerated by the scalp acupuncture treatment. Moreover, expression of pro-apoptotic protein CHOP, phosphorylated-JNK, cleaved capases-3 and -9 also decreased after the scalp acupuncture. In conclusion, the present study showed that scalp acupuncture of vertex middle line and anterior oblique line of vertex temple may alleviate cerebral ischemic stroke by inhibiting ER stress-accelerated apoptosis.

Abstract 267: Vascular Smooth Muscle Cell-specific Deletion Of Protein Kinase R-like Endoplasmic Reticulum Kinase Attenuates Microbiome-enhanced Abdominal Aortic Aneurysm

Background: We have previously linked the microbiome-derived metabolite trimethylamine N-oxide (TMAO) to abdominal aortic aneurysm (AAA) in both associative human and mechanistic mouse studies. TMAO is reported to bind protein kinase R-like endoplasmic reticulum kinase (PERK), resulting in selective activation of the unfolded protein response (UPR). Concordantly, our previous RNA sequencing results indicate that TMAO augments PERK-mediated UPR pathways in the aneurysm wall. Thus, the current study will determine the functional role of TMAO-mediated PERK activation in aortic VSMCs to AAA pathogenesis. Methods & Results: Ldlr -/- mice with VSMC-specific deletion of Perk were produced by breeding hemizygous male mice expressing Cre under the control of the Transgelin promoter ( B6.Cg-TG(Tagln-cre)1Her/J , Jackson Labs) to female Perk floxed mice ( Eif2ak3 tm1.2Drc /J , Jackson Labs). Specific deletion of PERK in VSMCs was validated by western blot. Male mice were subjected to angiotensin II (AngII) infusion via osmotic mini pump (1,000 ng/kg/min; 28 days), while female mice underwent laparotomy and application of topical elastase (10 mg/mL porcine pancreatic elastase 5 minutes) to induce AAA. To augment circulating TMAO levels, mice were fed a high choline diet (0.2% total cholesterol + 1.2% choline supplementation) for one week prior to and throughout the study. Male mice with VSMC specific PERK knockout demonstrated significantly reduced AngII-induced aortic dilation (Cre+: 0.90 ± 0.048 mm; Cre-: 1.57 ± 0.156 mm; P = 0.001) and rupture induced death relative to control. Similarly, female mice with VSMC specific PERK knockout displayed significantly reduced aortic diameter (Cre+: 1.17 ± 0.069 mm; Cre-: 2.65 ± 0.432 mm; P = 0.002) and rupture induced mortality as compared to control. Additionally, male and female mice with VSMC specific PERK knockout had significantly greater Type I collagen and reduced macrophage accumulation compared to control. Conclusions: These results indicate that VSMC specific knockout of PERK blunts AAA formation in two independent murine models. Further studies are needed to elucidate the mechanism of PERK mediated ER stress in AAA, which may reveal novel therapeutic targets for AAA treatment.

Chemical chaperone 4-PBA mitigates TNF alpha mediated ER stress in hASM cells

Airway inflammation and pro-inflammatory cytokines such as tumor necrosis factor alpha (TNFα) underlie the pathophysiology of respiratory diseases, including asthma. Previously, we showed that TNFα selectively activates the inositol-requiring enzyme 1α (IRE1α)/X-box binding protein 1 spliced (XBP1s) ER stress pathway in human airway smooth muscle (hASM) cells. The ER stress pathway is activated by the accumulation of unfolded proteins in the ER. Accordingly, chemical chaperones such as 4-phenylbutyrate (4-PBA) may ameliorate ER stress activation. In the present study, we hypothesize that 4-PBA mitigates TNFα induced ER stress in hASM cells. hASM cells were isolated from bronchiolar tissue obtained from 5 donors with no history of current smoking or respiratory diseases. hASM cells were serum-deprived for 48 h, and phenotype confirmed by expression of smooth muscle actin and fusiform morphology. hASM cells from the same donor were then separated into 4 12-h treatment groups: 1) TNFα (20 ng/mL), 2) TNFα + 4-PBA (30 min pretreatment), and 3) untreated control. Protein expression and phosphorylation of IRE1α and XBP1s were determined by Western blot. Gene expression of XBP1s was analyzed by PCR. We found that TNFα induced IRE1α phosphorylation and XBP1 splicing, which were significantly reduced by pretreatment with 4-PBA. These results support our hypothesis and suggest that, in the presence of chemical chaperone 4-PBA, TNFα induced ER stress is significantly decreased. R01HL 157984 (GCS) 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.

Immunomodulatory therapy with glatiramer acetate reduces endoplasmic reticulum stress and mitochondrial dysfunction in experimental autoimmune encephalomyelitis

Endoplasmic reticulum (ER) stress and mitochondrial dysfunction are found in lesions of multiple sclerosis (MS) and animal models of MS such as experimental autoimmune encephalomyelitis (EAE), and may contribute to the neuronal loss that underlies permanent impairment. We investigated whether glatiramer acetate (GA) can reduce these changes in the spinal cords of chronic EAE mice by using routine histology, immunostaining, and electron microscopy. EAE spinal cord tissue exhibited increased inflammation, demyelination, mitochondrial dysfunction, ER stress, downregulation of NAD+ dependent pathways, and increased neuronal death. GA reversed these pathological changes, suggesting that immunomodulating therapy can indirectly induce neuroprotective effects in the CNS by mediating ER stress.

Nogo-A Mediated Endoplasmic Reticulum Stress During Myocardial Ischemic-Reperfusion Injury in Diabetic Rats.

Among the three isoforms encoded by neurite outgrowth inhibitor proteins has been intensely investigated as a central nervous system inhibitor. Although neurite outgrowth inhibitor protein-A (Nogo-A) expression is increased in plasma of patients who have experienced a coronary heart disease, its role in heart disease is not well elucidated. In this study, we discovered a significant increase in Nogo-A expression in diabetic myocardial ischemia reperfusion (MI/R) injury conditions. Accelerated Nogo-A and MI/R injury in diabetic rats was attenuated by tauroursodeoxycholic acid treatment and knockdown of Nogo-A per se is sufficient to decrease endoplasmic reticulum (ER) stress as well as prevents cardiomyocyte apoptosis. We hypothesized that decreased Nogo-A levels might reducing diabetic MI/R injury. Nogo-A interacted with C/EBP homologous protein, suggesting a role for Nogo-A in ER stress during diabetic MI/R. In conclusion, Nogo-A mediated ER stress plays a major role in diabetic MI/R injury, and pathologically altered Nogo-A expression mediates diabetic MI/R injury, suggesting Nogo-A as a novel target for the treatment of diabetic MI/R injury in clinical settings.

Modulation of SREBP Expression and Fatty Acid Levels by Bacteria-Induced ER Stress Is Mediated by Hemocyanin in Penaeid Shrimp

Many environmental and pathogenic insults induce endoplasmic reticulum (ER) stress in animals, especially in aquatic ecosystems, where these factors are crucial for life. In penaeid shrimp, pathogens and environmental stressors induce hemocyanin expression, but the involvement of hemocyanin in ER stress response is unknown. We demonstrate that in response to pathogenic bacteria (Vibrio parahaemolyticus and Streptococcus iniae), hemocyanin, ER stress proteins (Bip, Xbp1s, and Chop), and sterol regulatory element binding protein (SREBP) are induced to alter fatty acid levels in Penaeus vannamei. Interestingly, hemocyanin interacts with ER stress proteins to modulate SREBP expression, while ER stress inhibition with 4-Phenylbutyric acid or hemocyanin knockdown attenuates the expression of ER stress proteins, SREBP, and fatty acid levels. Contrarily, hemocyanin knockdown followed by tunicamycin treatment (ER stress activator) increased their expression. Thus, hemocyanin mediates ER stress during pathogen challenge, which consequently modulates SREBP to regulate the expression of downstream lipogenic genes and fatty acid levels. Our findings reveal a novel mechanism employed by penaeid shrimp to counteract pathogen-induced ER stress.

The UDPase ENTPD5 regulates ER stress-associated renal injury by mediating protein N-glycosylation

Impaired protein N-glycosylation leads to the endoplasmic reticulum (ER) stress, which triggers adaptive survival or maladaptive apoptosis in renal tubules in diabetic kidney disease (DKD). Therapeutic strategies targeting ER stress are promising for the treatment of DKD. Here, we report a previously unappreciated role played by ENTPD5 in alleviating renal injury by mediating ER stress. We found that ENTPD5 was highly expressed in normal renal tubules; however, ENTPD5 was dynamically expressed in the kidney and closely related to pathological DKD progression in both human patients and mouse models. Overexpression of ENTPD5 relieved ER stress in renal tubular cells, leading to compensatory cell proliferation that resulted in hypertrophy, while ENTPD5 knockdown aggravated ER stress to induce cell apoptosis, leading to renal tubular atrophy and interstitial fibrosis. Mechanistically, ENTPD5-regulated N-glycosylation of proteins in the ER to promote cell proliferation in the early stage of DKD, and continuous hyperglycemia activated the hexosamine biosynthesis pathway (HBP) to increase the level of UDP-GlcNAc, which driving a feedback mechanism that inhibited transcription factor SP1 activity to downregulate ENTPD5 expression in the late stage of DKD. This study was the first to demonstrate that ENTPD5 regulated renal tubule cell numbers through adaptive proliferation or apoptosis in the kidney by modulating the protein N-glycosylation rate in the ER, suggesting that ENTPD5 drives cell fate in response to metabolic stress and is a potential therapeutic target for renal diseases.