Puerarin Inhibits the PERK-eIF2α-ATF4-CHOP Pathway through Inactivating JAK2/STAT3 Signal in Pancreatic beta-Cells

2021 ◽  
pp. 1-16
Author(s):  
Xiaobo Hu ◽  
Tingting Duan ◽  
Zhuan Wu ◽  
Cifei Tang ◽  
Zhaohui Cao
Keyword(s):  
Er Stress ◽  
Pancreatic Beta Cells ◽  
Critical Role ◽  
Underlying Mechanism ◽  
Signal Pathway ◽  
Janus Kinase ◽  
Cell Counting ◽  
Initiation Factor ◽  
Min6 Cells ◽  
Insulinoma Cell

Type 1 diabetes (T1D) is an autoimmune and inflammatory disease with excessive loss of pancreatic islet [Formula: see text]-cells. Accumulating evidence indicated that endoplasmic reticulum (ER) stress played a critical role in [Formula: see text]-cells loss, leading to T1D. Therefore, promoting the survival of pancreatic [Formula: see text]cells would be beneficial for patients with T1D. Puerarin is a natural isoflavone that has been demonstrated to be able to decrease blood glucose in patients with T1D. However, it remains unknown whether puerarin improves ER stress to prevent [Formula: see text]-cells from apoptosis. Here, we sought to investigate the role of puerarin in ER stress-associated apoptosis and explore its underlying mechanism in the mouse insulinoma cell line (MIN6). Flow cytometry and cell counting kit-8 (CCK8) experiments showed that puerarin caused a significant increase in the viability of MIN6 cells injured by H2O2. Furthermore, the protein kinase R-like ER kinase (PERK) signal pathway, a critical branch of ER stress response, was found to be involved in this process. Puerarin inhibited the phosphorylation of PERK, subsequently suppressed the phosphorylation of eukaryotic initiation factor 2[Formula: see text] (eIF2[Formula: see text], then decreased the activating transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP) expression, ultimately attenuating ER stress to prevent MIN6 cells from apoptosis. In addition, puerarin inhibited the activation of Janus kinase 2 (JAK2)/signal transducer and activators of transcription 3 (STAT3), which suppressed the PERK signal cascade with decreased ATF4 and CHOP levels. Taken together, our results firstly demonstrated that puerarin could prevent MIN6 cells from apoptosis at least in part by inhibiting the PERK-eIF2[Formula: see text]-ATF4-CHOP axis under ER stress conditions, which might be mediated by inactivation of the JAK2/STAT3 signal pathway. Therefore, investigating the mechanism underlying the effects of puerarin might highlight the potential roles of puerarin developing into an antidiabetic drug.

1,25-(OH)2D3 protects pancreatic beta cells against H2O2-induced apoptosis through inhibiting the PERK-ATF4-CHOP pathway

2020 ◽  
Author(s):  
Xiaobo Hu ◽  
Cong Hu ◽  
Jun Liu ◽  
Zhuan Wu ◽  
Tingting Duan ◽  
...  
Keyword(s):  
Er Stress ◽  
Cell Apoptosis ◽  
Pancreatic Beta Cells ◽  
Critical Role ◽  
Active Form ◽  
Cell Counting ◽  
Pancreatic Β Cell ◽  
Β Cell ◽  
Min6 Cells ◽  
Cell Destruction

Abstract Endoplasmic reticulum (ER) stress plays a critical role in pancreatic β cell destruction which leads to the pathogenesis of type 1 diabetes mellitus (T1DM). Vitamin D (VD) has been reported to reduce the risk of T1DM; however, it remains unknown whether VD affects ER stress in pancreatic β cells. In this study, we investigated the role of the active form of VD, 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3), in ER stress-induced β cell apoptosis and explored its potential mechanism in mouse insulinoma cell line mouse insulinoma 6 (MIN6). The results of cell counting kit-8 (CCK8) and flow cytometric analyses showed that 1,25-(OH)2D3 caused a significant increase in the viability of MIN6 cells injured by H2O2. The protein kinase like ER kinase (PERK) signal pathway, one of the most conserved branches of ER stress, was found to be involved in this process. H2O2 activated the phosphorylation of PERK, upregulated the activating transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP) expression, and subsequently initiated cell apoptosis, which were significantly reversed by 1,25-(OH)2D3 pretreatment. In addition, GSK2606414, a specific inhibitor of PERK, suppressed PERK phosphorylation and reduced the expressions of ATF4 and CHOP, leading to a significant decrease in β cell apoptosis induced by H2O2. Taken together, the present findings firstly demonstrated that 1,25-(OH)2D3 could prevent MIN6 cells against ER stress-associated apoptosis by inhibiting the PERK-ATF4-CHOP pathway. Therefore, our results suggested that 1,25-(OH)2D3 might serve as a potential therapeutic target for preventing pancreatic β cell destruction in T1DM.

scholarly journals Eukaryotic translation initiation factor 2A protects pancreatic beta cells during endoplasmic reticulum stress while rescuing translation inhibition

2021 ◽  
Author(s):  
Evgeniy Panzhinskiy ◽  
Søs Skovsø ◽  
Haoning Cen ◽  
Kwan Chu ◽  
Kate MacDonald ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Er Stress ◽  
Protein Translation ◽  
Human Islets ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Related Factor ◽  
Min6 Cells ◽  
Primary Mouse ◽  
Mouse Islets

Abstract The endoplasmic reticulum (ER) stress-induced unfolded protein response (UPR) helps decide cell survival in diabetes. The alternative eukaryotic initiation factor 2A (EIF2A) has been proposed to mediate EIF2S1-independent translation during cellular stress and viral infection, but its role in cells is unknown. EIF2A abundance is high in human and mouse islets relative to other tissues, and both thapsigargin and palmitate significantly increased EIF2A mRNA and EIF2A protein levels in MIN6 cells, mouse islets and human islets. Knockdowns of EIF2A, the related factor EIF2D, or both EIF2A and EIF2D, were not sufficient to cause apoptosis. On the other hand, transient or stable EIF2A over-expression protected MIN6 cells, primary mouse islets, and human islets from ER stress-induced, caspase-3-dependent apoptosis. Mechanistically, EIF2A overexpression decreased ERN1 (also known as IRE1) expression in thapsigargin-treated MIN6 cells or human islets. In vivo, cell specific EIF2A viral overexpression reduced ER stress, improved insulin secretion, and abrogated hyperglycemia in Ins2Akita/WT mice. EIF2A overexpression significantly increased expression of genes involved in protein translation and reduced expression of pro-apoptotic genes (e.g. ALDH1A3). Remarkably, the decrease in global protein synthesis during UPR was prevented by EIF2A, despite ER stress-induced EIF2S1 phosphorylation. The protective effects of EIF2A were additive to those of ISRIB, a drug that counteracts the effects of EIF2S1 phosphorylation. Cells overexpressing EIF2A showed higher expression of translation factor EIF2B5, which may contribute to the lack of translational inhibition in these cells. We conclude that EIF2A is a novel target for cell protection and the circumvention of EIF2S1-mediated translational repression.

A novel secretagogin/ATF4 pathway is involved in oxidized LDL-induced endoplasmic reticulum stress and islet β-cell apoptosis

2020 ◽  
Author(s):  
Li Wu ◽  
Yuncheng Lv ◽  
Ying Lv ◽  
Sunmin Xiang ◽  
Zhibo Zhao ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Transcription Factor ◽  
Er Stress ◽  
Cell Apoptosis ◽  
Initiation Factor ◽  
Β Cells ◽  
Β Cell ◽  
Immunoprecipitation Assay ◽  
Min6 Cells ◽  
Stress Markers

Abstract Excessive accumulation of cholesterol in β cells initiates endoplasmic reticulum (ER) stress and associated apoptosis. We have reported that excessive uptake of cholesterol by MIN6 cells decreases the expression of secretagogin (SCGN) and then attenuates insulin secretion. Here, we aimed to determine whether cholesterol-induced SCGN decrease is involved in the modulation of ER stress and apoptosis in pancreatic β cells. In this study, MIN6 cells were treated with oxidized low-density lipoprotein (ox-LDL) for 24 h, and then intracellular lipid droplets and cell apoptosis were quantified, and SCGN and ER stress markers were identified by western blot analysis. Furthermore, small interfer RNA (siRNA)-mediated SCGN knockdown and recombinant plasmid-mediated SCGN restoration experiments were performed to confirm the role of SCGN in ER stress and associated cell apoptosis. Finally, the interaction of SCGN with ATF4 was computationally predicted and then validated by a co-immunoprecipitation assay. We found that ox-LDL treatment increased the levels of ER stress markers, such as phosphorylated protein kinase-like endoplasmic reticulum kinase, phosphorylated eukaryotic initiation factor 2 alpha, activating transcription factor 4 (ATF4), and transcription factor CCAAT-enhancer-binding protein homologous protein, and promoted MIN6 cell apoptosis; in addition, the expression of SCGN was downregulated. siRNA-mediated SCGN knockdown exacerbated β-cell ER stress by increasing ATF4 expression. Pretreatment of MIN6 cells with the recombinant SCGN partly antagonized ox-LDL-induced ER stress and apoptosis. Furthermore, a co-immunoprecipitation assay revealed an interaction between SCGN and ATF4 in MIN6 cells. Taken together, these results demonstrated that pancreatic β-cell apoptosis induced by ox-LDL treatment can be attributed, in part, to an SCGN/ATF4-dependent ER stress response.

scholarly journals Eukaryotic translation initiation factor 2A protects pancreatic beta cells during endoplasmic reticulum stress while rescuing translation inhibition

2021 ◽  
Author(s):  
Evgeniy Panzhinskiy ◽  
Søs Skovsø ◽  
Haoning Howard Cen ◽  
Kwan Yi Chu ◽  
Kate MacDonald ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Er Stress ◽  
Protein Translation ◽  
Human Islets ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Β Cell ◽  
Min6 Cells ◽  
Primary Mouse ◽  
Mouse Islets

ABSTRACTThe endoplasmic reticulum (ER) stress-induced unfolded protein response (UPR) helps decide β cell survival in diabetes. The alternative eukaryotic initiation factor 2A (EIF2A) has been proposed to mediate EIF2S1-independent translation during cellular stress and viral infection, but its role in β cells is unknown. EIF2A abundance is high in human and mouse islets relative to other tissues, and both thapsigargin and palmitate significantly increased EIF2A mRNA and EIF2A protein levels in MIN6 cells, mouse islets and human islets. Knockdowns of EIF2A, the related factor EIF2D, or both EIF2A and EIF2D, were not sufficient to cause apoptosis. On the other hand, transient or stable EIF2A over-expression protected MIN6 cells, primary mouse islets, and human islets from ER stress-induced, caspase-3-dependent apoptosis. Mechanistically, EIF2A overexpression decreased ERN1 (also known as IRE1α) expression in thapsigargin-treated MIN6 cells or human islets. In vivo, β cell specific EIF2A viral overexpression reduced ER stress, improved insulin secretion, and abrogated hyperglycemia in Ins2Akita/WT mice. EIF2A overexpression significantly increased expression of genes involved in protein translation and reduced expression of pro-apoptotic genes (e.g. ALDH1A3). Remarkably, the decrease in global protein synthesis during UPR was prevented by EIF2A, despite ER stress-induced EIF2S1 phosphorylation. The protective effects of EIF2A were additive to those of ISRIB, a drug that counteracts the effects of EIF2S1 phosphorylation. Cells overexpressing EIF2A showed higher expression of translation factor EIF2B5, which may contribute to the lack of translational inhibition in these cells. We conclude that EIF2A is a novel target for β cell protection and the circumvention of EIF2S1-mediated translational repression.

scholarly journals Knockdown of long noncoding RNA HOTAIR inhibits osteoarthritis chondrocyte injury by miR-107/CXCL12 axis

2021 ◽  
Vol 16 (1) ◽  
Author(s):  
Jipeng Lu ◽  
Zhongxiong Wu ◽  
Ying Xiong
Keyword(s):  
Cell Proliferation ◽  
Western Blot ◽  
Cell Apoptosis ◽  
Noncoding Rna ◽  
Underlying Mechanism ◽  
Joint Disease ◽  
Cell Counting ◽  
Luciferase Reporter ◽  
Cxcl12 Expression ◽  
Ecm Degradation

Abstract Background Osteoarthritis (OA) is a joint disease characterized via destruction of cartilage. Chondrocyte damage is associated with cartilage destruction during OA. Long noncoding RNAs (lncRNAs) are implicated in the regulation of chondrocyte damage in OA progression. This study aims to investigate the role and underlying mechanism of lncRNA homeobox antisense intergenic RNA (HOTAIR) in OA chondrocyte injury. Methods Twenty-three OA patients and healthy controls without OA were recruited. Chondrocytes were isolated from OA cartilage tissues. HOTAIR, microRNA-107 (miR-107) and C-X-C motif chemokine ligand 12 (CXCL12) levels were measured by quantitative real-time polymerase chain reaction and western blot. Cell proliferation, apoptosis and extracellular matrix (ECM) degradation were measured using cell counting kit-8, flow cytometry and western blot. The target interaction was explored by bioinformatics, luciferase reporter and RNA immunoprecipitation assays. Results HOTAIR expression was enhanced, and miR-107 level was reduced in OA cartilage samples. HOTAIR overexpression inhibited cell proliferation, but induced cell apoptosis and ECM degradation in chondrocytes. HOTAIR knockdown caused an opposite effect. MiR-107 was sponged and inhibited via HOTAIR, and knockdown of miR-107 mitigated the effect of HOTAIR silence on chondrocyte injury. CXCL12 was targeted by miR-107. CXCL12 overexpression attenuated the roles of miR-107 overexpression or HOTAIR knockdown in the proliferation, apoptosis and ECM degradation. CXCL12 expression was decreased by HOTAIR silence, and restored by knockdown of miR-107. Conclusion HOTAIR knockdown promoted chondrocyte proliferation, but inhibited cell apoptosis and ECM degradation in OA chondrocytes by regulating the miR-107/CXCL12 axis.

scholarly journals Palmitate Causes Endoplasmic Reticulum Stress and Apoptosis in Human Mesenchymal Stem Cells: Prevention by AMPK Activator

Endocrinology ◽  
2012 ◽  
Vol 153 (11) ◽  
pp. 5275-5284 ◽  
Author(s):  
Jun Lu ◽  
Qinghua Wang ◽  
Lianghu Huang ◽  
Huiyue Dong ◽  
Lingjing Lin ◽  
...  
Keyword(s):  
Stem Cells ◽  
Endoplasmic Reticulum ◽  
Mesenchymal Stem Cells ◽  
Protein Kinase ◽  
Er Stress ◽  
Human Mesenchymal Stem Cells ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Human Umbilical Cord ◽  
Amp Activated Protein Kinase

Abstract Elevated circulating saturated fatty acids concentration is commonly associated with poorly controlled diabetes. The highly prevalent free fatty acid palmitate could induce apoptosis in various cell types, but little is known about its effects on human mesenchymal stem cells (MSCs). Here, we report that prolonged exposure to palmitate induces human bone marrow-derived MSC (hBM-MSC) and human umbilical cord-derived MSC apoptosis. We investigated the role of endoplasmic reticulum (ER) stress, which is known to promote cell apoptosis. Palmitate activated XBP1 splicing, elF2α (eukaryotic translation initiation factor 2α) phosphorylation, and CHOP, ATF4, BiP, and GRP94 transcription in hBM-MSCs. ERK1/2 and p38 MAPK phosphorylation were also induced by palmitate in hBM-MSCs. A selective p38 inhibitor inhibited palmitate activation of the ER stress, whereas the ERK1/2 inhibitors had no effect. The AMP-activated protein kinase activator aminoimidazole carboxamide ribonucleotide blocked palmitate-induced ER stress and apoptosis. These findings suggest that palmitate induces ER stress and ERK1/2 and p38 activation in hBM-MSCs, and AMP-activated protein kinase activator prevents the deleterious effects of palmitate by inhibiting ER stress and apoptosis.

Identification of spatial specifically expressed genes in rice (Oryza sativa) seedlings using cDNA microarray

2004 ◽  
Vol 1 (2) ◽  
pp. 133-139
Author(s):  
Sun Liang-Xian ◽  
Dong Hai-Tao ◽  
Zhuang Xiao-Feng ◽  
Zhang Feng ◽  
Li De-Bao
Keyword(s):  
Ribosomal Protein ◽  
Lipid Transfer Protein ◽  
Protein Biosynthesis ◽  
Critical Role ◽  
Phosphoglycerate Kinase ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Metabolic Reaction ◽  
Lipid Transfer ◽  
Molecular Physiology

AbstractMembranous cDNA microarrays containing 2200 unique rice transcripts were designed for screening the characteristics of spatially expressed genes in post-germination rice seedlings. By comparing the profiles obtained, 31 genes were identified as expressed specifically in the plumule, 36 in the mesocotyl and 73 in the radicle. Several genes, such as polyubiquitin, UDP-glucose pyrophosphorylase, sucrose synthase and phosphoglycerate kinase, which encode components of the carbohydrate or protein metabolic reaction cascades, were expressed specifically in the mesocotyl, indicating that degradation reactions of the endospermous reserve starch and proteins occur mainly in the mesocotyl during the post-germination stage. A number of genes involved in defence mechanisms or in the processes of replication, transcription and translation were identified as expressed specifically in the plumule or radicle. Among plumule specifically expressed genes, translation initiation factor 5a, 40s ribosomal protein s28 and ribosomal protein 136 are considered to have a critical role in protein biosynthesis; while allergenic protein, β-D-glucan exohydrolase and actin 11 are genes with defending functions. Among the catalogue of radicle specifically expressed genes, EF-1a, Tat binding protein, replication protein A2, histone h3.2, ribosomal protein s29a and 40s ribosomal protein s19 are genes that function in the process of replication, transcription or translation; whereas glycine-rich protein, wound-induced basic protein, Bowman-Birk proteinase inhibitor and lipid transfer protein-2 are genes involved in defence responses. Results of this experiment have provided insight into post-germination molecular physiology at the genomic level of gene expression.

scholarly journals Mammalian ECD Protein Is a Novel Negative Regulator of the PERK Arm of the Unfolded Protein Response

2017 ◽  
Vol 37 (18) ◽  
Author(s):  
Appolinaire A. Olou ◽  
Aniruddha Sarkar ◽  
Aditya Bele ◽  
C. B. Gurumurthy ◽  
Riyaz A. Mir ◽  
...  
Keyword(s):  
Er Stress ◽  
Translation Initiation Factor ◽  
Negative Regulator ◽  
Initiation Factor ◽  
Mrna Levels ◽  
Produce Cell ◽  
Stress Induction ◽  
Content Type ◽  
Protein Levels ◽  
The Unfolded Protein Response

ABSTRACT Mammalian Ecdysoneless (ECD) is a highly conserved ortholog of the Drosophila Ecd gene product whose mutations impair the synthesis of Ecdysone and produce cell-autonomous survival defects, but the mechanisms by which ECD functions are largely unknown. Here we present evidence that ECD regulates the endoplasmic reticulum (ER) stress response. ER stress induction led to a reduced ECD protein level, but this effect was not seen in PKR-like ER kinase knockout (PERK-KO) or phosphodeficient eukaryotic translation initiation factor 2α (eIF2α) mouse embryonic fibroblasts (MEFs); moreover, ECD mRNA levels were increased, suggesting impaired ECD translation as the mechanism for reduced protein levels. ECD colocalizes and coimmunoprecipitates with PERK and GRP78. ECD depletion increased the levels of both phospho-PERK (p-PERK) and p-eIF2α, and these effects were enhanced upon ER stress induction. Reciprocally, overexpression of ECD led to marked decreases in p-PERK, p-eIF2α, and ATF4 levels but robust increases in GRP78 protein levels. However, GRP78 mRNA levels were unchanged, suggesting a posttranscriptional event. Knockdown of GRP78 reversed the attenuating effect of ECD overexpression on PERK signaling. Significantly, overexpression of ECD provided a survival advantage to cells upon ER stress induction. Taken together, our data demonstrate that ECD promotes survival upon ER stress by increasing GRP78 protein levels to enhance the adaptive folding protein in the ER to attenuate PERK signaling.

Differential activation of ER stress and apoptosis in response to chronically elevated free fatty acids in pancreatic β-cells

2008 ◽  
Vol 294 (3) ◽  
pp. E540-E550 ◽  
Author(s):  
Elida Lai ◽  
George Bikopoulos ◽  
Michael B. Wheeler ◽  
Maria Rozakis-Adcock ◽  
Allen Volchuk
Keyword(s):  
Er Stress ◽  
Cell Lines ◽  
Cell Apoptosis ◽  
Human Islets ◽  
Pancreatic Β Cell ◽  
Β Cell ◽  
Min6 Cells ◽  
Relative Contribution ◽  
Induced Apoptosis

Chronic exposure to elevated saturated free fatty acid (FFA) levels has been shown to induce endoplasmic reticulum (ER) stress that may contribute to promoting pancreatic β-cell apoptosis. Here, we compared the effects of FFAs on apoptosis and ER stress in human islets and two pancreatic β-cell lines, rat INS-1 and mouse MIN6 cells. Isolated human islets cultured in vitro underwent apoptosis, and markers of ER stress pathways were elevated by chronic palmitate exposure. Palmitate also induced apoptosis in MIN6 and INS-1 cells, although the former were more resistant to both apoptosis and ER stress. MIN6 cells were found to express significantly higher levels of ER chaperone proteins than INS-1 cells, which likely accounts for the ER stress resistance. We attempted to determine the relative contribution that ER stress plays in palmitate-induced β-cell apoptosis. Although overexpressing GRP78 in INS-1 cells partially reduced susceptibility to thapsigargin, this failed to reduce palmitate-induced ER stress or apoptosis. In INS-1 cells, palmitate induced apoptosis at concentrations that did not result in significant ER stress. Finally, MIN6 cells depleted of GRP78 were more susceptible to tunicamycin-induced apoptosis but not to palmitate-induced apoptosis compared with control cells. These results suggest that ER stress is likely not the main mechanism involved in palmitate-induced apoptosis in β-cell lines. Human islets and MIN6 cells were found to express high levels of stearoyl-CoA desaturase-1 compared with INS-1 cells, which may account for the decreased susceptibility of these cells to the cytotoxic effects of palmitate.

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