Mesenchymal stem cells reduce ER stress via PERK-Nrf2 pathway in an aged mouse model

이은주,( Nayra Cárdenes ),( Diana Álvarez ),( Jacobo Sellares ),( John Sembrat ),( Paola Aranda ),( Yating Peng ),( Jordan Bullock ),( Seyed Mehdi Nouraie ),( Ana L Mora ),( Mauricio Rojas ) 대한결핵 및 호흡기학회 2018 대한결핵 및 호흡기학회 추계학술대회 초록집 Vol.126 No.-

Mesenchymal stem cells (MSCs) have been shown to ameliorate the deleterious effects of bleomycin. However, the mechanism responsible for protection of stem cell therapy in pulmonary fibrosis is still poorly understood, especially in terms of the endoplasmic reticulum (ER) stress. We hypothesized that ER stress could increase after a certain time elapse and then decrease to the baseline, resembling the fibrosis after bleomycin exposure. We also attempted to clarify the exact role of MSCs upon ER stress by a timely sacrifice of animals at the peak of ER stress. In order to determine the peak time in ER stress, the expression of ER stress marker proteins after bleomycin injury were measured in the lung of old mice at different time points (day 0, 3, 7, 14, and 21). For evaluation of the effect of MSCs on ER stress, we assessed the bleomycin insult by changes in body weight, lung tissues histology and the protein expression of ER stress markers. The level of expression of unfolded protein response (UPR) transcription factor XBP-1 and its regulator BiP were elevated at day 7 and progressively increased up to day 21. MSCs inhibited BiP expression in bleomycin-induced ER stress, whereas the expression levels of other ER stress markers were not reduced by MSCs. MSCs operate on ER stress via several pathways, but the PERK-Nrf2 pathway revealed to be the main functioning pathway. In conclusion, we demonstrated that MSCs attenuate ER stress via PERK-Nrf2 pathway.

A Role of GADD153 in ER Stress-induced Apoptosis in Recombinant Chinese Hamster Ovary Cells

Chaya Mohan,Madhavi Sathyamurthy,이균민 한국생물공학회 2012 Biotechnology and Bioprocess Engineering Vol.17 No.3

The imbalance between the folding capacity and the folding demand imposed on the endoplasmic reticulum (ER) of therapeutic protein-producing host cells results in a stressed ER. This initiates a series of cellular signaling events termed the unfolded protein response (UPR) aimed at restoring homeostasis. In order to alleviate ER stress and ER stress-induced apoptosis in recombinant Chinese hamster ovary (rCHO) cells, silencing of the growth arrest and DNA damage 153 gene (GADD153), the main pro-apoptotic factor of UPR, was attempted. The rCHO cells were cultured under four ER stress inducing conditions, including thapsigargin, brefeldin A, glucose deprivation, glucose and glutamine deprivation. In these conditions, the functions of stably GADD153-silenced clones were investigated. It was found that under exclusive ER stress-inducing conditions of thapsigargin and brefeldin A treatments, the GADD153-silenced clones showed a less incidence of apoptosis (about 38%) and less cell viability (about 58% non-viable cells) than the control cells. However, under nutrient deprivation, the beneficial effect of GADD153 silencing was not pronounced because nutrient deprivation led to a cascade of various events including GADD153-induced cell death. GADD153-overexpressing pool cells also substantiated the findings of GADD153 downregulation. Investigation of the underlying mechanism revealed that increased GADD153 expression results in an exaggerated production of reactive oxygen species (ROS) and that GADD153 silencing promotes translational attenuation facilitating cell recovery from stress. Taken together, this study suggests that GADD153 sensitizes cells to ER stress through mechanisms that involve enhanced oxidative injury and by manipulating the ER client protein load in rCHO cells. The imbalance between the folding capacity and the folding demand imposed on the endoplasmic reticulum (ER) of therapeutic protein-producing host cells results in a stressed ER. This initiates a series of cellular signaling events termed the unfolded protein response (UPR) aimed at restoring homeostasis. In order to alleviate ER stress and ER stress-induced apoptosis in recombinant Chinese hamster ovary (rCHO) cells, silencing of the growth arrest and DNA damage 153 gene (GADD153), the main pro-apoptotic factor of UPR, was attempted. The rCHO cells were cultured under four ER stress inducing conditions, including thapsigargin, brefeldin A, glucose deprivation, glucose and glutamine deprivation. In these conditions, the functions of stably GADD153-silenced clones were investigated. It was found that under exclusive ER stress-inducing conditions of thapsigargin and brefeldin A treatments, the GADD153-silenced clones showed a less incidence of apoptosis (about 38%) and less cell viability (about 58% non-viable cells) than the control cells. However, under nutrient deprivation, the beneficial effect of GADD153 silencing was not pronounced because nutrient deprivation led to a cascade of various events including GADD153-induced cell death. GADD153-overexpressing pool cells also substantiated the findings of GADD153 downregulation. Investigation of the underlying mechanism revealed that increased GADD153 expression results in an exaggerated production of reactive oxygen species (ROS) and that GADD153 silencing promotes translational attenuation facilitating cell recovery from stress. Taken together, this study suggests that GADD153 sensitizes cells to ER stress through mechanisms that involve enhanced oxidative injury and by manipulating the ER client protein load in rCHO cells.

재조합 단백질 생산을 위한 소포체 신호전달

구태원,윤은영,강석우,권기상,권오유,Goo, Tae-Won,Yun, Eun-Young,Kang, Seok-Woo,Kwon, Ki-Sang,Kwon, O-Yu 한국생명과학회 2007 생명과학회지 Vol.17 No.6

ER-Golgi 분비 경로를 통해서 정확한 구조를 가지면서 post-translational modification 과정을 거친 재조합 단백질의 발현을 최대화하는 것은 ER stress반응에 대한 연구의 중요한 계기가 된다. 세포가 스트레스를 받지 않는 상태라도 ER stress signaling은 재조합 단백질의 생산량을 제한하고 품질을 떨어뜨리는 여러 가지 조건을 만들게 된다. ER stress signaling을 막는 여러 가지 방법들이 제시되고 있으며 표 2는 이러한 방법들 중 일부를 나타내고 있다. 일반적으로는 pro-survival 경로에 관련되어 있는 인자를 촉진하고 pro-apoptosis에 관련되어 있는 인자를 억제하는 것들이다. 그러나 ER stress 반응은 매우 복잡하고 적응과 사멸 기작(adaptation and elimination mechanism)의 중간 역할을 하기 때문에 ER stress에 관련된 주요 인자를 산업적으로 응용하기 위해선 이들의 기능에 대해 보다 깊은 연구가 이루어져야 한다. 현재까지 재조합단백질의 생산량을 최대한으로 높이는 방법은 ER stress 반응이 생기지 않도록 fed-batch process를 개선하고 세포 사멸 기작을 조절하며 단백질의 glycosylation 처리를 하는 것이다. The endoplasmic reticulum (ER) is an important intracellular organelle for folding and maturation of newly synthesized transmembrane and secretory proteins. The ER provides stringent quality control systems to ensure that only correctly folded proteins exit the ER and unfolded or misfolded proteins are retained and ultimately degraded. The ER has evolved stress response both signaling pathways the unfolded protein response (UPR) to cope with the accmulation of unfolded or misfolded proteins and ER overload response (EOR). Accumulating evidence suggests that, in addition to responsibility for protein processing, ER is also an important signaling compartment and a sensor of cellular stress. In this respect, production of bio-functional recombinant-proteins requires efficient functioning of the ER secretory pathway in host cells. This review briefly summarizes our understanding of the ER signaling developed in the recent years to help of the secretion capacities of recombinant cells.

ER Stress-Induced Jpk Expression and the Concomitant Cell Death

Kim, Hye-Sun,Chung, Hyun-Joo,Kong, Kyoung-Ah,Park, Sung-Do,Kim, Myoung-Hee 대한의생명과학회 2005 Biomedical Science Letters Vol.11 No.2

A Jopock (Jpk), a trans-acting factor associating with the position-specific regulatory element of murine Hoxa-7, has shown to have a toxicity to both prokaryotic and eukaryotic cells when over expressed. Since Jpk protein harbors a trans membrane domain and a putative endoplasmic reticulum (ER)-retention signal at the N-terminus, a sub cellular localization of the protein was analyzed after fusing it into the green fluorescent protein (GFP): Both N-term (Jpk-EGFP) and C-term tagged-Jpk (EGFP-Jpk) showed to be localized in the ER when analyzed under the fluorescence microscopy after staining the cells with ER- and Mito Tracker. Since ER stress triggers the ER-stress mediated apoptosis to eliminate the damaged cells, we analyzed the expression pattern of Jpk under ER-stress condition. When MCF7 cells were treated with the ER-stress inducer such as DTT and EGTA, the expression of Jpk was upregulated at the transcriptional level like that of Grp78, a molecular chaperone well known to be over expressed under ER-stress condition. In the presence of high concentration of ER-sterss inducer (10 mM), about 70 (DTT) to 95% (EGTA) of cells died stronly expressing (10~12 fold) Jpk. Whereas at the low concentration (0.001~1.0 mM) of the inducer, the expression of Jpk was increased about 2.5 (EGTA) to 5 fold (DTT), which is rather similar to those of ER chaperone protein Grp78. These results altogether indicate that the ER-stress upregulated the expression of Jpk and the excess stress induces the ER-stress induced apoptosis and the concomitant expression of Jpk.

Endoplasmic recticulum stress와 관련된 유전자기능과 전사조절인자의 in silico 분석

김태민(Tae-Min Kim),여지영(Jiyoung Yeo),박찬선(Chan Sun Park),이문수(Moon Soo Rhee),정명호(Myeong Ho Jung) 한국생명과학회 2009 생명과학회지 Vol.19 No.8

ER stress에 관련된 유전자의 기능변화와 전사조절인자 분석하기 위해 ER stress를 유도한 간세포에서 expression microarray로 유전자 발현을 확보한 후 GSECA로 분석하였다. ER stress가 유도되면, ER에 주어지는 과도한 부하를 감소시키는 기능들이 증가하는 반면, ER stress가 더 증가함에 따라 ATP 생성이나 DNA repair, 더 나아가 세포분열의 기능이 감소하는 등 세포가 damage을 받음을 알 수 있었다. ER stress에 관련된 전사조절인자로는 FOXO4, AP-1, FOXO3, HNF4, IRF-1, GATA 등의 전사조절인자들이 ER stress에 의해 발현이 증가하는 유전자들의 promoter에 공통적으로 존재하였으며, E2F, Nrf-1, Elk-1, YY1, CREB, MTF-1, STAT-1, ATF 등의 전사인자 들이 발현이 감소하는 유전자들의 promoter에서 공통적으로 존재하여, 이들의 전사인자들이 ER stress에 의한 유전자의 발현조절에 중요한 역할을 하는 전사조절인자임을 알 수 있었다. It has been postulated that endoplasmic (ER) stress is involved in the development of several diseases. However, the detailed molecular mechanisms have not been fully understood. Therefore, we characterized a genetic network of genes induced by ER stress using cDNA microarray and gene set expression coherence analysis (GSECA), and identified gene function as well as several transcription regulators associated with ER stress. We analyzed time-dependent gene expression profiles in thapsigargin-treated Sk-Hep1 using an oligonucleotide expression chip, and then selected functional gene sets with significantly high expression coherence which was processed into functional clusters according to the expression similarities. The functions related to sugar binding, lysosome, ribosomal protein, ER lumen, and ER to golgi transport increased, whereas the functions with mRNA processing, DNA replication, DNA repair, cell cycle, electron transport chain and helicase activity decreased. Furthermore, functional clusters were investigated for the enrichment of regulatory motifs using GSECA, and several transcriptional regulators associated with regulation of ER-induced gene expression were found.

SREBP-1c Ablation Protects Against ER Stress-induced Hepatic Steatosis by Preventing Impaired Fatty Acid Oxidation

Young-Seung Lee(이영승),Timothy F. Osborne(티모씨 에프 오스본),Young-Kyo Seo(서영교),Tae-Il Jeon(전태일) 한국생명과학회 2021 생명과학회지 Vol.31 No.9

간 소포체(ER) 스트레스는 비알콜성지방간과 인슐린 저항성의 발달에 기여하고, unfolded protein response(UPR)의 구성요소는 지질 대사를 조절한다. 최근 연구에 따르면 ER 스트레스와 비정상적인 세포 지질 대사 사이의 연관성이 보고되었으며, 이 과정에서 지질 대사의 중심 조절자인 sterol regulatory element binding proteins(SREBPs)의 관련성이 확인되었다. 그러나 ER 스트레스 동안 지질 대사를 조절하는 SREBP의 정확한 역할과 비알콜성지방간에 대한 기여는 아직 밝혀지지 않았다. 본 연구에서 SREBP-1c 결핍은 UPR, 염증 및 지방산 산화 조절을 통해 ER 스트레스에 의해 유도된 비알콜성지방간으로부터 생쥐를 보호한다는 것을 보여준다. SREBP-1c는 inositol requiring kinase 1α (IRE1α) 발현을 직접적으로 조절하고 ER 스트레스에 의해 유도된 tumor necrosis factor-α의 활성화를 매개하여 peroxisome proliferator-activated receptor γ coactivator 1-α (PGC1α)의 감소와 그에 따른 지방산 산화의 장애를 유발한다. 그러나, SREBP-1c의 유전적 결핍은 이러한 현상을 보호하여 간 염증과 지방축적을 완화시킨다. SREBP-1c 결핍이 ER 스트레스에 의해 유도된 염증 신호를 방지하는 메커니즘은 아직 밝혀지지 않았지만, SREBP-1c가 결핍된 Kupffer 세포에서 IRE1α 신호의 변화가 염증 신호에 관여할 수 있을 것으로 생각된다. 본 연구결과는 SREBP-1c가 ER 스트레스에 의해 유도된 비알콜성지방간에서 UPR 및 염증의 조절에 중요한 역할을 함을 시사한다. Hepatic endoplasmic reticulum (ER) stress contributes to the development of steatosis and insulin resistance. The components of unfolded protein response (UPR) regulate lipid metabolism. Recent studies have reported an association between ER stress and aberrant cellular lipid control; moreover, research has confirmed the involvement of sterol regulatory element-binding proteins (SREBPs)-the central regulators of lipid metabolism-in the process. However, the exact role of SREBPs in controlling lipid metabolism during ER stress and its contribution to fatty liver disease remain unknown. Here, we show that SREBP-1c deficiency protects against ER stress-induced hepatic steatosis in mice by regulating UPR, inflammation, and fatty acid oxidation. SREBP-1c directly regulated inositol-requiring kinase 1α (IRE1α) expression and mediated ER stress-induced tumor necrosis factor-α activation, leading to a reduction in expression of peroxisome proliferator-activated receptor γ coactivator 1-α and subsequent impairment of fatty acid oxidation. However, the genetic ablation of SREBP-1c prevented these events, alleviating hepatic inflammation and steatosis. Although the mechanism by which SREBP-1c deficiency prevents ER stress-induced inflammatory signaling remains to be elucidated, alteration of the IRE1α signal in SREBP-1c-depleted Kupffer cells might be involved in the signaling. Overall, the results suggest that SREBP-1c plays a crucial role in the regulation of UPR and inflammation in ER stress-induced hepatic steatosis.

4-Phenylbutyric acid reduces mutant-TGFBIp levels and ER stress through activation of ERAD pathway in corneal fibroblasts of granular corneal dystrophy type 2

Choi, S.i.,Lee, E.,Jeong, J.B.,Akuzum, B.,Maeng, Y.S.,Kim, T.i.,Kim, E.K. Academic Press 2016 Biochemical and biophysical research communication Vol.477 No.4

Granular corneal dystrophy type 2 (GCD2) is caused by a point mutation (R124H) in the transforming growth factor β-induced (TGFBI) gene. In GCD2 corneal fibroblasts, secretion of the accumulated mutant TGFBI-encoded protein (TGFBIp) is delayed via the endoplasmic reticulum (ER)/Golgi-dependent secretory pathway. However, ER stress as the pathogenic mechanism underlying GCD2 has not been fully characterized. The aim of this study was to confirm whether ER stress is linked to GCD2 pathogenesis and whether the chemical chaperone, 4-phenylbutyric acid (4-PBA), could be exploited as a therapy for GCD2. We found that the ER chaperone binding immunoglobulin protein (BiP) and the protein disulfide isomerase (PDI) were elevated in GCD2. Western bolt analysis also showed a significant increase in both the protein levels and the phosphorylation of the key ER stress kinases, inositol-requiring enzyme 1α (IRE1α) and double stranded RNA activated protein kinase (PKR)-like ER kinase, as well as in levels of their downstream targets, X box-binding protein 1 (XBP1) and activating transcription factor 4, respectively, in GCD2 corneal fibroblasts. GCD2 cells were found to be more susceptible to ER stress-induced cell death than were wild-type corneal fibroblasts. Treatment with 4-PBA considerably reduced the levels of BiP, IRE1α, and XBP1 in GCD2 cells; notably, 4-PBA treatment significantly reduced the levels of TGFBIp without change in TGFBI mRNA levels. In addition, TGFBIp levels were significantly reduced under ER stress and this reduction was considerably suppressed by the ubiquitin proteasome inhibitor MG132, indicating TGFBIp degradation via the ER-associated degradation pathway. Treatment with 4-PBA not only protected against the GCD2 cell death induced by ER stress but also significantly suppressed the MG132-mediated increase in TGFBIp levels under ER stress. Together, these results suggest that ER stress might comprise an important factor in GCD2 pathophysiology and that the effects of 4-PBA treatment might have important implications for the development of GCD2 therapeutics.

Enhanced PRL-1 in Placenta-derived Mesenchymal Stem Cells Promotes Liver Regeneration in Cirrhotic Rat Model via Regulating ER Stress-Dependent Calcium Homeostasis

( Se Ho Kim ),( Jae Yeon Kim ),( Soo Young Park ),( Ji Hye Jun ),( Hee Jung Park ),( Jin Man Kim ),( Gi Jin Kim ) 대한간학회 2020 춘·추계 학술대회 (KASL) Vol.2020 No.1

Aims: ER stress in liver is caused by the accumulation of cholesterol and perturbation of calcium in ER. In our previous report, placenta-derived mesenchymal stem cells (PD-MSCs) have the therapeutic potential of hepatic regeneration in cirrhosis rat model. However, whether phosphatase of regenerating liver 1 (PRL-1) is related ER stress remains unknown. Here, we demonstrate that PRL-1 genetically modified PD-MSCs (PD-MSCs^PRL-1) promote hepatic functions, regulating ER stress and calcium release in rat model with bile duct ligation (BDL). Methods: Naïve and PD-MSCs^PRL-1 (2x10⁶) were intravenously transplanted into BDL rat model. WB-F344s (rat liver epithelial cell) exposed to thapsigargin (TG) were co-cultured with naïve and PD-MSCs^PRL-1. The therapeutic effects were analyzed by qPCR, western blotting, immunohistochemistry (IHC), and imaging using calcium biosensor. Results: ER stress markers were increased in BDL model (NTx; non-transplantation), while PD-MSCs^PRL-1 transplantation (Tx PD-MSCs^PRL-1) showed significantly decrease compared to naïve (Tx; Naïve PD-MSCs). PD-MSCs^PRL-1 induced changes of voltage-dependent channels related gene expressions compared to naïve. In addition, PD-MSCs^RPL-1 inhibited the expression of apoptosis related markers, while increased proliferation activity of hepatocyte using IHC with PCNA. In vitro TG treated WBF344, ER stress induced by depletion of calcium in ER. PD-MSCs^PRL-1 co-culture significantly decreased the expression of ER stress markers compared to naïve. Interestingly, PD-MSCs^PRL-1 co-culture induced calcium influx by dynamic changes through ER and cytoplasm-specific calcium homeostasis compared to naïve (P<0.05). Also, decreased ER stress by PD-MSC^PRL-1 enhanced hepatic regeneration. Conclusions: PD-MSCs^PRL-1 was involved in calcium homeostasis via decreased ER stress in cirrhotic rat model. Therefore, PD-MSCs^PRL-1 could be new strategy in degenerative medicine including hepatic diseases. Funding: This research was supported by a grant of the Ministry of Health & Welfare, Republic of Korea (HI17C1050) and by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2020M3A9B302618221).

Endoplasmic Reticulum Stress Activates Hepatic Macrophages through PERK-hnRNPA1 Signaling

Kwon Ari,Kim Yun Seok,Kim Jiyoon,Koo Ja Hyun 한국응용약물학회 2024 Biomolecules & Therapeutics(구 응용약물학회지) Vol.32 No.3

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.

Inhibition of Endoplasmic Reticulum Stress-induced Apoptosis by Silkworm Storage Protein 1

차연주,백지은,이원종 한국생물공학회 2018 Biotechnology and Bioprocess Engineering Vol.23 No.2

The endoplasmic reticulum (ER) plays essential roles indispensable for cellular activity and survival, including functions such as protein synthesis, secretory and membrane protein folding, and Ca2+ release in cells. The ER is sensitive to stresses that can lead to the aggregation and accumulation of misfolded proteins, which eventually triggers cellular dysfunction; severe or prolonged ER stress eventually induces apoptosis. ER stress-induced apoptosis causes several devastating diseases such as atherosclerosis, neurodegenerative diseases, and diabetes. In addition, the production of biopharmaceuticals such as monoclonal antibodies requires the maintenance of normal ER functions to achieve and maintain the production of high-quality products in good quantities. Therefore, it is necessary to develop methods to efficiently relieve ER stress and protect cells from ER stress-induced apoptosis. The silkworm storage protein 1 (SP1) has anti-apoptotic activities that inhibit the intrinsic mitochondrial apoptotic pathway. However, the role of SP1 in controlling ER stress and ER stress-induced apoptosis has not been investigated. In this paper, we demonstrate that SP1 can inhibit apoptosis induced by a well-known ER stress inducer, thapsigargin, by alleviating the decrease in cell viability and mitochondrial membrane potential. Interestingly, SP1 significantly blocked increases in CHOP and GRP78 expression as well as ER Ca2+ leakage into the cytosol following ER stress induction. This indicates that SP1 protects cells from ER stressinduced apoptosis by functioning as an upstream inhibitor of apoptosis. Therefore, studying SP1 function can offer new insights into protecting cells against ER stress-induced apoptosis for future applications in the biopharmaceutical and medicine industries.