Regulation Of Endoplasmic Reticulum Stress Response Research Articles

Endoplasmic Reticulum Stress Activates Hepatic Macrophages through PERK-hnRNPA1 Signaling.

Endoplasmic reticulum (ER) stress plays a crucial role in liver diseases, affecting various types of hepatic cells. While studies have focused on the link between ER stress and hepatocytes as well as hepatic stellate cells (HSCs), the precise involvement of hepatic macrophages in ER stress-induced liver injury remains poorly understood. Here, we examined the effects of ER stress on hepatic macrophages and their role in liver injury. Acute ER stress led to the accumulation and activation of hepatic macrophages, which preceded hepatocyte apoptosis. Notably, macrophage depletion mitigated liver injury induced by ER stress, underscoring their detrimental role. Mechanistic studies revealed that ER stress stimulates macrophages predominantly via the PERK signaling pathway, regardless of its canonical substrate ATF4. hnRNPA1 has been identified as a crucial mediator of PERK-driven macrophage activation, as the overexpression of hnRNPA1 effectively reduced ER stress and suppressed pro-inflammatory activation. We observed that hnRNPA1 interacts with mRNAs that encode UPR-related proteins, indicating its role in the regulation of ER stress response in macrophages. These findings illuminate the cell type-specific responses to ER stress and the significance of hepatic macrophages in ER stress-induced liver injury. Collectively, the PERK-hnRNPA1 axis has been discovered as a molecular mechanism for macrophage activation, presenting prospective therapeutic targets for inflammatory hepatic diseases such as acute liver injury.

Identification of Key Endoplasmic Reticulum Stress-Related Genes in Non-Alcoholic Fatty Liver Disease

Background: Endoplasmic reticulum stress (ERS) is involved in the etiology of non-alcoholic fatty liver disease (NAFLD). Thus, the current study was designed to identify key ERS-associated genes in NAFLD. Methods: RNA-Seq data of NAFLD and controls were sourced from the Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) in NAFLD and controls were identified by limma. By overlapping DEGs and ERS-related genes, ERS-related DEGs were identified. The function of ERS-related DEGs was characterized by clusterProfiler. Next, the protein–protein interaction (PPI) network was created using the Cytoscape software and the STRING database to identify key ERS-related genes in NAFLD. Furthermore, the correlations among key ERS-related genes were calculated. Results: A total of 8965 DEGs were identified between NAFLD and controls in the GSE126848 dataset. After overlapping these DEGs and ERS-related genes, 20 genes were identified as ERS-related DEGs in NAFLD. Functional analysis revealed that the genes mainly participated in ER-related functions, such as the ER–nucleus signaling pathway, regulation of ERS response, and protein processing in ER. The PPI network revealed the interactions among 17 ERS-related DEGs, including ERN1, ATF6, and EIF2S1 as the key genes. The expressions of ERN1, ATF6, and EIF2S1 were significantly down-regulated in NAFLD and were strongly positively correlated with each other. Further, the expression of ERN1 and ATFA6 was also similar in the GSE89632 datasets. Conclusion: The present study identified ERN1, ATF6, and EIF2S1 as key ERS-related genes in NAFLD. These findings may provide a molecular basis for the role of ERS in NAFLD.

Effects of melatonin on neural reconstruction after acute spinal cord injury through regulation of endoplasmic reticulum stress response and autophagy

It has been reported that autophagy and endoplasmic reticulum (ER) stress response cause improved hindlimb motor function and reduced damage of axons in spinal cord injured animals, respectively. However, the effects of melatonin on neural reconstruction and motor recovery through regulation of ER stress response and autophagy have not been well described. Therefore, the purpose of this study is to elucidate the effects of melatonin treatment on neural reconstruction and motor recovery through regulation of ER stress response and autophagy.To verify the effect of melatonin injection on post‐SCI alterations regarding neural cells, autophagy, and ER stress response, we analyzed markers at protein level, and morphological changes. At day 3 after SCI, melatonin did not cause behavioral improvement (p<.05). At molecular levels, melatonin suppressed the loss of astrocytes after SCI (p<.05). Beclin‐1 expression was decreased by melatonin treatment (p<.05). In other words, autophagy activation in SCI+MT was lower than that of SCI group, indicating melatonin‐mediated suppression of autophagic cell death. In addition, GRP78 expression was preserved in SCI+MT group with no difference compared to non‐injured groups. Namely, ER stress response was advanced by melatonin. In histological analysis, it was proved that dendritic branches were preserved by melatonin treatment.In conclusion, exogenous treatment of melatonin may result in neural reconstruction after SCI through regulating autophagy and ER stress response at the injured spinal segments.Support or Funding InformationNRF‐2017R1A2A2A01067169, KGM4611821, 2016 Creative Research Program of Inje University, Republic of KoreaThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A novel role of c-FLIP protein in regulation of ER stress response

Cellular-Flice-like inhibitory protein (c-FLIP) is an apoptosis modulator known to inhibit the extrinsic apoptotic pathway thus blocking Caspase-8 processing in the Death Inducing Signalling Complex (DISC). We previously demonstrated that c-FLIP localizes at the endoplasmic reticulum (ER) and that c-FLIP-deficient mouse embryonic fibroblasts (MEFs) display an enlarged ER morphology. In the present study, we have addressed the consequences of c-FLIP ablation in the ER stress response by investigating the effects of pharmacologically-induced ER stress in Wild Type (WT) and c-FLIP−/− MEFs. Surprisingly, c-FLIP−/− MEFs were found to be strikingly more resistant than WT MEFs to ER stress-mediated apoptosis. Analysis of Unfolded Protein Response (UPR) pathways revealed that Pancreatic ER Kinase (PERK) and Inositol-Requiring Enzyme 1 (IRE1) branch signalling is compromised in c-FLIP−/− cells when compared with WT cells. We found that c-FLIP modulates the PERK pathway by interfering with the activity of the serine threonine kinase AKT. Indeed, c-FLIP−/− MEFs display higher levels of active AKT than WT MEFs upon ER stress, while treatment with a specific AKT inhibitor of c-FLIP−/− MEFs subjected to ER stress restores the PERK but not the IRE1 pathway. Importantly, the AKT inhibitor or dominant negative AKT transfection sensitizes c-FLIP−/− cells to ER stress-induced cell death while the expression of a constitutively active AKT reduces WT cells sensitivity to ER stress-induced death. Thus, our results demonstrate that c-FLIP modulation of AKT activity is crucial in controlling PERK signalling and sensitivity to ER stress, and highlight c-FLIP as a novel molecular player in PERK and IRE1-mediated ER stress response.

A novel role of c-FLIP protein in regulation of ER stress response

Cellular-Flice-like Inhibitory Protein (c-FLIP) is an apoptosis modulator known to inhibit the extrinsic apoptotic pathway thus blocking Caspase-8 processing in the Death Inducing Signalling Complex (DISC). We previously demonstrated that c-FLIP localizes at the Endoplasmic Reticulum (ER) and that c-FLIP-deficient Mouse Embryonic Fibroblasts (MEFs) display an enlarged ER morphology. In the present study, we have addressed the consequences of c-FLIP ablation in the ER stress response by investigating the effects of pharmacologically-induced ER stress in Wild Type (WT) and c-FLIP-/- MEFs. Surprisingly, c-FLIP-/- MEFs were found to be strikingly more resistant than WT MEFs to ER stress-mediated apoptosis. Analysis of Unfolded Protein Response (UPR) pathways revealed that Pancreatic ER Kinase (PERK) and Inositol-Requiring Enzyme 1 (IRE1) branch signalling is compromised in c-FLIP-/- cells when compared with WT cells. We found that c-FLIP modulates the PERK pathway by interfering with the activity of the serine threonine kinase AKT. Indeed, c-FLIP-/- MEFs display higher levels of active AKT than WT MEFs upon ER stress, while treatment with a specific AKT inhibitor of c-FLIP-/- MEFs subjected to ER stress restores the PERK but not the IRE1 pathway. Importantly, the AKT inhibitor or dominant negative AKT transfection sensitizes c-FLIP-/- cells to ER stress-induced cell death while the expression of a constitutively active AKT reduces WT cells sensitivity to ER stress-induced death. Thus, our results demonstrate that c-FLIP modulation of AKT activity is crucial in controlling PERK signalling and sensitivity to ER stress, and highlight c-FLIP as a novel molecular player in PERK and IRE1-mediated ER stress response.

Abstract 4959: Klotho suppresses colon cancer through modulation of the Wnt pathway and unfolded protein response

Abstract Aberrant activation of the canonical Wnt signal transduction pathway is implicated in the development and progression of colorectal cancer (CC). The key effector protein of this pathway is β-catenin, which functions with T-cell factor/lymphoid enhancer factor (TCF/LEF) to activate expression of Wnt target genes. Klotho is a transmembrane protein, which can be cleaved, shed and act as a circulating hormone. Klotho-deficient mice manifest a syndrome resembling accelerated aging, while klotho overexpression extends life span. We have previously identified klotho as a potent tumor suppressor in breast and pancreatic cancer, and recent data indicate it as a potent tumor suppressor in a wide array of malignancies, including CC. The activities of klotho include inhibition of the insulin like growth factor (IGF)-1 and the Wnt pathways. We aimed to decipher the role of klotho as a tumor suppressor and modulator of the Wnt pathway in CC. We first tested the effect of klotho on proliferation of CC cells and noted that overexpression of klotho inhibited colony formation of the HCT 116, SW480, HT-29, Colo-320 and RKO CC cells. We next examined the effects of klotho in vivo, using the azoxymethane (AOM) model, and found that daily administration of klotho to mice inhibited formation of colon polyps induced by AOM. While we did not see an effect of klotho on the IGF-1 signaling pathway, klotho did affect the Wnt pathway. Klotho reduced Wnt3A and β-catenin protein levels as measured by Western blotting, and inhibited transcriptional activity of the Wnt pathway as evidenced in luciferase assay. Moreover, this transcriptional inhibition was abrogated by overexpression of a constitutively active β-catenin. We, therefore, suspected that klotho inhibited the pathway upstream of β-catenin. Indeed, co-immunoprecipitation analyses indicated direct interaction between klotho and Wnt3A. As the inhibitory effect of klotho on colony formation of CC cells was only partially rescued by transfection with constitutively active β-catenin, we concluded that additional mechanisms may mediate the effects of klotho. In order to elucidate these mechanisms, we conducted a cDNA expression microarray and involvement of klotho in endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) was observed. Taken together, our data indicate klotho as a potent tumor suppressor in CC and suggest its role already at the early stages of polyp formation. Klotho effects in CC are partially mediated by the Wnt pathway however additional mechanisms, including regulation of ER stress response, are involved. Citation Format: Tammi Rubinstein, Shiri Shahmoon, Gil Har Zahav, Nir Skalka, Tal Etan, Rina Arbesfeld, Metsada Pasmanik-Chor, Tami Rubinek, Ido Wolf. Klotho suppresses colon cancer through modulation of the Wnt pathway and unfolded protein response. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4959. doi:10.1158/1538-7445.AM2015-4959

Effect of Altered Endogenous Melatonin Regulated by Conditional Light Exposure on Neural Reorganization by Regulation of ER Stress Response and Autophagy after Spinal Cord Injury

Spinal cord injury causes unfolded protein response (UPR) to recover from endoplasmic reticulum (ER) stress. Melatonin is known to delay ER stress‐induced apoptosis. Furthermore, it has been suggested that melatonin‐mediated autophagy can induce neuroprotection.The purpose of this study was to elucidate the effect of endogenous melatonin on neural reorganization and locomotor recovery after spinal cord injury. To diversify the concentration of endogenous melatonin, differentiated light exposure time was needed. Thus, experimental animals were divided into 5 groups; group1 (12‐hr light/12‐hr dark), group2 (6‐hr light/18‐hr dark), group3 (24‐hr dark), group4 (18‐hr light/6‐hr dark), group5 (24‐hr light). 7 days after injury, animals were engaged in locomotor assessment.The BBB score of experimental animals was increased inversely proportional to light exposure time. Similarly, the more the rats were blocked from light, the more neural cells were reorganized. Furthermore, axonal sprouting showed a tendency to relatively increase in accordance with light blockade.In conclusion, UPR and autophagy might interact to play a neuroprotective role and, this effect is intensified by endogenous melatonin.Funding : 2012R1A1A2005089, 2013R1A2A2A01067169, KGM4611512, 2014R1A1A3051724

Effects of Melatonin on Neural Reconstruction and Motor Recovery through Regulation of Endoplasmic Reticulum Stress Response and Autophagy in Spinal Cord Injured Animal

Autophagy and endoplasmic reticulum (ER) stress response are known to cause improved motor function and reduced damage of axons after spinal cord injury. This study aims to elucidate the effects of melatonin treatment on neural reconstruction and motor recovery through regulation of ER stress response and autophagy.
 Expression of neural and autophagic markers at mRNA level increased in injured experimental animals. And much higher increase of those markers was observed when treated with melatonin. Especially, at day 3, 28 after SCI, NG2 expression was increased at the injured site when treated with melatonin (p<.05). With melatonin treatment, LC3 expression was also increased at day 28 after surgery (p<.05). There was a tendency for spinal cord injured groups and melatonin‐treated groups to show increased ER stress response markers, when compared with the expression of non‐injured groups and non‐melatonin‐treated groups, respectively. Furthermore, more axonal sprouting and relatively intact morphology were observed in melatonin‐treated groups. Locomotor function was also improved when treated with melatonin.
 In conclusion, melatonin treatment promoted the neural reconstruction of both neuronal and glial cells. It also reduced autophagic cell death and, consequently brought improved locomotor function.Funding : 2012R1A1A2005089, 2013R1A2A2A01067169, KGM4611512, 2014R1A1A3051724

Progranulin is required for proper ER stress response and inhibits ER stress-mediated apoptosis through TNFR2

Progranulin (PGRN) was reported to be a stress-response factor in response to hypoxia and acidosis. Here we present evidences demonstrating that PGRN is also an endoplasmic reticulum (ER) stress responsive factor: PGRN expression was induced and its activation of Erk1/2 and Akt signaling enhanced in response to ER stress; Normal ER stress response was lost in PGRN deficient cells and PGRN deficient cells became hypersusceptible to ER stress-induced apoptosis; additionally, recombinant PGRN could rescue the defects in ER-stress responses seen in PGRN deficient cells. Mechanistic studies indicated that PGRN/TNFR2 was critical for PGRN mediated regulation of ER stress response: similar to PGRN, the expression of TNFR2, but not TNFR1, was also induced in the course of ER stress; in addition, the association between PGRN and TNFR2 was markedly enhanced following ER stress; More importantly, PGRN protection of ER stress induced apoptosis was abolished when TNFR2 signaling was blocked. In addition, the 2nd and 3rd cysteine-rich domains (CRD) in the extracellular portion of TNFR2 (CRD2CRD3), known to directly bind to PGRN, disturbed the interaction of PGRN with TNFR2, and in turn abolished PGRN-mediated activation of Erk1/2 and Akt signaling and protection against apoptosis in response to ER-stress. Collectively, PGRN plays an important role in ER stress and regulates ER stress response through interacting with TNFR2. This study provides new insight into PGRN regulation of stress response and may also present PGRN as a potential molecular target for treating stress-associated disorders.

Management of the endoplasmic reticulum stress by activation of the heat shock response in yeast

In yeast Saccharomyces cerevisiae, accumulation of misfolded proteins in the endoplasmic reticulum (ER) causes ER stress and activates the unfolded protein response (UPR), which is mediated by Hac1p. The heat shock response (HSR) mediated by Hsf1p, mainly regulates cytosolic processes and protects the cell from stresses. Here, we find that a constitutive activation of the HSR could increase ER stress resistance in both wild-type and UPR-deficient cells. Activation of HSR decreased UPR activation in the WT (as shown by the decreased HAC1 mRNA splicing). We analyzed the genome-wide transcriptional response in order to propose regulatory mechanisms that govern the interplay between UPR and HSR and followed up for the hypotheses by experiments in vivo and in vitro. Interestingly, we found that the regulation of ER stress response via HSR is (1) only partially dependent on over-expression of Kar2p (ER resident chaperone induced by ER stress); (2) does not involve the increase in protein turnover via the proteasome activity; (3) is related to the oxidative stress response. From the transcription data, we also propose that HSR enhances ER stress resistance mainly through facilitation of protein folding and secretion. We also find that HSR coordinates multiple stress-response pathways, including the repression of the overall transcription and translation.

Abstract 454: Endoplasmic Reticulum Stress Response in Pressure-Overloaded Ischemic-Reperfused Hearts

An integral component of endoplasmic reticulum (ER) stress-induced apoptosis is expression of growth arrest- and DNA damage inducible protein 153 (GADD153); this protein is normally expressed in low levels but its expression markedly increases following sustained stress to the ER. GADD153 regulates both apoptosis and inflammatory response. We previously showed that pressure overload exacerbates myocardial ischemia reperfusion (IR) injury. In this study, we tested the hypothesis that pressure overload regulates ER stress response manifested as increased GADD153 expression thereby upregulating inflammatory cytokine production and contributing to worsening of myocardial IR injury. Accordingly, Langendorff-perfused rat hearts were subjected to global IR protocol with perfusion pressure set at 80 or 160 cmH 2 O; normoxic hearts served as controls. Compared to normoxia, an IR insult increased expressions of pro-inflammatory cytokine (interleukin (IL)-17) and GADD153 in association with increased cell death. In the ischemic-reperfused hearts, pressure overload a) increased expression of GADD153, b) reduced interleukin (IL)-10 but increased IL-17 and c) increased annexin V immunostaining as well as apoptotic and necrotic cell death. Collectively, the results suggest that pressure overload exacerbates cell death in the isolated ischemic-reperfused heart involving regulation of ER stress response and inflammation.

Enhanced Lysosomal Activity Is Involved in Bax Inhibitor-1-induced Regulation of the Endoplasmic Reticulum (ER) Stress Response and Cell Death against ER Stress: INVOLVEMENT OF VACUOLAR H+-ATPASE (V-ATPASE)

Bax inhibitor-1 (BI-1) is an evolutionarily conserved protein that protects cells against endoplasmic reticulum (ER) stress while also affecting the ER stress response. In this study, we examined BI-1-induced regulation of the ER stress response as well as the control of the protein over cell death under ER stress. In BI-1-overexpressing cells (BI-1 cells), proteasome activity was similar to that of control cells; however, the lysosomal fraction of BI-1 cells showed sensitivity to degradation of BSA. In addition, areas and polygonal lengths of lysosomes were greater in BI-1 cells than in control cells, as assessed by fluorescence and electron microscopy. In BI-1 cells, lysosomal pH was lower than in control cells and lysosomal vacuolar H(+)-ATPase(V-ATPase), a proton pump, was activated, suggesting high H(+) uptake into lysosomes. Even when exposed to ER stress, BI-1 cells maintained high levels of lysosomal activities, including V-ATPase activity. Bafilomycin, a V-ATPase inhibitor, leads to the reversal of BI-1-induced regulation of ER stress response and cell death due to ER stress. In BI-1 knock-out mouse embryo fibroblasts, lysosomal activity and number per cell were relatively lower than in BI-1 wild-type cells. This study suggests that highly maintained lysosomal activity may be one of the mechanisms by which BI-1 exerts its regulatory effects on the ER stress response and cell death.