Manganese activates autophagy to alleviate endoplasmic reticulum stress–induced apoptosis via PERK pathway

Overexposure to manganese (Mn) is neurotoxic. Our previous research has demonstrated that the interaction of endoplasmic reticulum (ER) stress and autophagy participates in the early stage of Mn‐mediated neurotoxicity in mouse. However, the mechanisms of ER stress signalling pathways in the initiation of autophagy remain confused. In the current study, we first validated that ER stress–mediated cell apoptosis is accompanied by autophagy in SH‐SY5Y cells. Then, we found that inhibiting ER stress with 4‐phenylbutyrate (4‐PBA) decreased ER stress–related protein expression and reduced cell apoptosis, whereas blocking autophagy with 3‐methyladenine (3‐MA) increased cell apoptosis. These data indicate that protective autophagy was activated to alleviate ER stress–mediated apoptosis. Knockdown of the protein kinase RNA‐like ER kinase (PERK) gene inhibited Mn‐induced autophagy and weakened the interaction between ATF4 and the LC3 promoter. Our results reveal a novel molecular mechanism in which ER stress may regulate autophagy via the PERK/eIF2α/ATF4 signalling pathway. Additionally, Mn may activate protective autophagy to alleviate ER stress–mediated apoptosis via the PERK/eIF2α/ATF4 signalling pathway in SH‐SY5Y cells.     

6-Shogaol induces apoptosis in human hepatocellular carcinoma cells and exhibits anti-tumor activity in vivo through endoplasmic reticulum stress

6-Shogaol is an active compound isolated from Ginger (Zingiber officinale Rosc). In this work, we demonstrated that 6-shogaol induces apoptosis in human hepatocellular carcinoma cells in relation to caspase activation and endoplasmic reticulum (ER) stress signaling. Proteomic analysis revealed that ER stress was accompanied by 6-shogaol-induced apoptosis in hepatocellular carcinoma cells. 6-shogaol affected the ER stress signaling by regulating unfolded protein response (UPR) sensor PERK and its downstream target eIF2α. However, the effect on the other two UPR sensors IRE1 and ATF6 was not obvious. In prolonged ER stress, 6-shogaol inhibited the phosphorylation of eIF2α and triggered apoptosis in SMMC-7721 cells. Salubrinal, an activator of the PERK/eIF2α pathway, strikingly enhanced the phosphorylation of eIF2α in SMMC-7721 cells with no toxicity. However, combined treatment with 6-shogaol and salubrinal resulted in significantly increase of apoptosis and dephosphorylation of eIF2α. Overexpression of eIF2α prevented 6-shogaol-mediated apoptosis in SMMC-7721 cells, whereas inhibition of eIF2α by small interfering RNA markedly enhanced 6-shogaol-mediated cell death. Furthermore, 6-shogaol-mediated inhibition of tumor growth of mouse SMMC-7721 xenograft was associated with induction of apoptosis, activation of caspase-3, and inactivation of eIF2α. Altogether our results indicate that the PERK/eIF2α pathway plays an important role in 6-shogaol-mediated ER stress and apoptosis in SMMC-7721 cells in vitro and in vivo.     

Inhibition of TGFβ‐activated protein kinase 1 ameliorates myocardial ischaemia/reperfusion injury via endoplasmic reticulum stress suppression

Transforming growth factor β‐activated protein kinase 1 (TAK1) involves in various biological responses and is a key regulator of cell death. However, the role of TAK1 on acute myocardial ischaemia/reperfusion (MI/R) injury is unknown. We observed that TAK1 activation increased significantly after MI/R and hypoxia/reoxygenation (H/R), and we hypothesized that TAK1 has an important role in MI/R injury. Mice (TAK1 inhibiting by 5Z‐7‐oxozeaenol or silencing by AAV9 vector) were exposed to MI/R injury. Primary cardiomyocytes (TAK1 silencing by siRNA; and overexpressing TAK1 by adenovirus vector) were used to induce H/R injury model in vitro. Inhibition of TAK1 significantly decreased MI/R‐induced myocardial infarction area, reduced cell death and improved cardiac function. Mechanistically, TAK1 silencing suppressed MI/R‐induced myocardial oxidative stress and attenuated endoplasmic reticulum (ER) stress both in vitro and in vivo. In addition, the inhibition of ROS by NAC partially reversed the damage of TAK1 in vitro. Our study presents the first direct evidence that inhibition of TAK1 mitigated MI/R injury, and TAK1 mediated ROS/ER stress/apoptosis signal pathway is important for the pathogenesis of MI/R injury.     

Modulation of the eukaryotic initiation factor 2 alpha-subunit kinase PERK by tyrosine phosphorylation

The endoplasmic reticulum (ER)-resident protein kinase PERK attenuates protein synthesis in response to ER stress through the phosphorylation of translation initiation factor eIF2alpha at serine 51. ER stress induces PERK autophosphorylation at several serine/threonine residues, a process that is required for kinase activation and phosphorylation of eIF2alpha. Herein, we demonstrate that PERK also possesses tyrosine kinase activity. Specifically, we show that PERK is capable of autophosphorylating on tyrosine residues in vitro and in vivo. We further show that tyrosine 615, which is embedded in a highly conserved region of the kinase domain of PERK, is essential for autocatalytic activity. That is, mutation of Tyr-615 to phenylalanine compromises the autophosphorylation capacity of PERK and the phosphorylation of eIF2alpha in vitro and in vivo. The Y615F mutation also impairs the ability of PERK to induce translation of ATF4. Immunoblot analyses with a phosphospecific antibody confirm the phosphorylation of PERK at Tyr-615 both in vitro and in vivo. Thus, our data classify PERK as a dual specificity kinase whose regulation by tyrosine phosphorylation contributes to its optimal activation in response to ER stress.     

Sulodexide attenuates endoplasmic reticulum stress induced by myocardial ischaemia/reperfusion by activating the PI3K/Akt pathway

Acute myocardial ischaemia/reperfusion (MI/R) injury causes severe arrhythmias with a high rate of lethality. Extensive research focus on endoplasmic reticulum (ER) stress and its dysfunction which leads to cardiac injury in MI/R Our study evaluated the effects of sulodexide (SDX) on MI/R by establishing MI/R mice models and in vitro oxidative stress models in H9C2 cells. We found that SDX decreases cardiac injury during ischaemia reperfusion and decreased myocardial apoptosis and infarct area, which was paralleled by increased superoxide dismutase and reduced malondialdehyde in mice plasm, increased Bcl‐2 expression, decreased BAX expression in a mouse model of MI/R. In vitro, SDX exerted a protective effect by the suppression of the ER stress which induced by tert‐butyl hydroperoxide (TBHP) treatment. Both of the in vivo and in vitro effects were involved in the phosphatidylinositol 3‐kinase (PI3K)/Akt signalling pathway. Inhibition of PI3K/Akt pathway by specific inhibitor, LY294002, partially reduced the protective effect of SDX. In short, our results suggested that the cardioprotective role of SDX was related to the suppression of ER stress in mice MI/R models and TBHP‐induced H9C2 cell injury which was through the PI3K/Akt signalling pathway.     

Mfn2 modulates the UPR and mitochondrial function via repression of PERK

Mitofusin 2 (Mfn2) is a key protein in mitochondrial fusion and it participates in the bridging of mitochondria to the endoplasmic reticulum (ER). Recent data indicate that Mfn2 ablation leads to ER stress. Here we report on the mechanisms by which Mfn2 modulates cellular responses to ER stress. Induction of ER stress in Mfn2‐deficient cells caused massive ER expansion and excessive activation of all three Unfolded Protein Response (UPR) branches (PERK, XBP‐1, and ATF6). In spite of an enhanced UPR, these cells showed reduced activation of apoptosis and autophagy during ER stress. Silencing of PERK increased the apoptosis of Mfn2‐ablated cells in response to ER stress. XBP‐1 loss‐of‐function ameliorated autophagic activity of these cells upon ER stress. Mfn2 physically interacts with PERK, and Mfn2‐ablated cells showed sustained activation of this protein kinase under basal conditions. Unexpectedly, PERK silencing in these cells reduced ROS production, normalized mitochondrial calcium, and improved mitochondrial morphology. In summary, our data indicate that Mfn2 is an upstream modulator of PERK. Furthermore, Mfn2 loss‐of‐function reveals that PERK is a key regulator of mitochondrial morphology and function.     

Lycopene Protects against Hypoxia/Reoxygenation Injury by Alleviating ER Stress Induced Apoptosis in Neonatal Mouse Cardiomyocytes

Endoplasmic reticulum (ER) stress induced apoptosis plays a pivotal role in myocardial ischemia/reperfusion (I/R)-injury. Inhibiting ER stress is a major therapeutic target/strategy in treating cardiovascular diseases. Our previous studies revealed that lycopene exhibits great pharmacological potential in protecting against the I/R-injury in vitro and vivo, but whether attenuation of ER stress (and) or ER stress-induced apoptosis contributes to the effects remains unclear. In the present study, using neonatal mouse cardiomyocytes to establish an in vitro model of hypoxia/reoxygenation (H/R) to mimic myocardium I/R in vivo, we aimed to explore the hypothesis that lycopene could alleviate the ER stress and ER stress-induced apoptosis in H/R-injury. We observed that lycopene alleviated the H/R injury as revealed by improving cell viability and reducing apoptosis, suppressed reactive oxygen species (ROS) generation and improved the phosphorylated AMPK expression, attenuated ER stress as evidenced by decreasing the expression of GRP78, ATF6 mRNA, sXbp-1 mRNA, eIF2α mRNA and eIF2α phosphorylation, alleviated ER stress-induced apoptosis as manifested by reducing CHOP/GADD153 expression, the ratio of Bax/Bcl-2, caspase-12 and caspase-3 activity in H/R-treated cardiomyocytes. Thapsigargin (TG) is a potent ER stress inducer and used to elicit ER stress of cardiomyocytes. Our results showed that lycopene was able to prevent TG-induced ER stress as reflected by attenuating the protein expression of GRP78 and CHOP/GADD153 compared to TG group, significantly improve TG-caused a loss of cell viability and decrease apoptosis in TG-treated cardiomyocytes. These results suggest that the protective effects of lycopene on H/R-injury are, at least in part, through alleviating ER stress and ER stress-induced apoptosis in neonatal mouse cardiomyocytes.     

Saturated fatty acid induction of endoplasmic reticulum stress and apoptosis in human liver cells via the PERK/ATF4/CHOP signaling pathway

Accumulation of saturated fatty acids in the liver can cause nonalcoholic fatty liver disease (NAFLD). This study investigated saturated fatty acid induction of endoplasmic reticulum (ER) stress and apoptosis in human liver cells and the underlying causal mechanism. Human liver L02 and HepG2 cell lines were exposed to the saturated fatty acid sodium palmitate. MTT assay was used for cell viability, flow cytometry and Hoechst 33258 staining for apoptosis, RT-PCR for mRNA expression, and Western blot for protein expression. Silence of PRK-like ER kinase (PERK) expression in liver cells was through transient transfection of PERK shRNA. Treatment of L02 and HepG2 cells with sodium palmitate reduced cell viability through induction of apoptosis. Sodium palmitate also induced ER stress in the cells, indicated by upregulation of PERK phosphorylation and expression of BiP, ATF4, and CHOP proteins. Sodium palmitate had little effect on activating XBP-1, a common target of the other two canonical sensors of ER stress, ATF6, and IRE1. Knockdown of PERK gene expression suppressed the PERK/ATF4/CHOP signaling pathway during sodium palmitate-induced ER stress and significantly inhibited sodium palmitate-induced apoptosis in L02 and HepG2 cells. Saturated fatty acid-induced ER stress and apoptosis in these human liver cells were enacted through the PERK/ATF4/CHOP signaling pathway. Future study is warranted to investigate the role of these proteins in mediating saturated fatty acid-induced NAFLD in animal models.     

PACE4对异丙肾上腺素诱导的心肌细胞凋亡的影响及其机制

目的:研究前体蛋白转化酶枯草溶菌素(PACE4)对异丙肾上腺素(ISO)诱导的心肌细胞凋亡的作用及其可能的作用机制。方法:构建pFLAG-PACE4重组表达载体并转染H9c2心肌细胞。将心肌细胞分为四组:正常对照组(无任何干预因素)、ISO组(10μmol/L ISO)、ISO+pFLAG组(空载质粒pFLAG转染+10μmol/LISO)、ISO+pFLAG-PACE4组(pFLAG-PACE4重组表达质粒转染+10μmol/LISO)。采用AnnexinV-FITC/PI双染法测定心肌细胞凋亡率;蛋白免疫印迹法检测活性半胱氨酸蛋白酶(caspase)-3、caspase-12、钙网蛋白、葡萄糖调节蛋白78(GRP78)、CCAAT/增强子结合蛋白同源蛋白(CHOP)、活化转录因子4(ATF4)和PERK的表达以及真核起始因子2α(eIF2α)的磷酸化水平。结果:与正常对照组相比,ISO组中PACE4表达水平明显降低,而转染pFLAG-PACE4质粒后,其表达水平显著增加。PACE4过表达可以显著抑制ISO诱导的细胞凋亡和caspase-3以及caspase-12的蛋白表达。ISO处理显著增加内质网应激分子钙网蛋白、GRP78和CHOP的表达,而PACE4过表达则可以抑制这些蛋白的表达。ISO诱导的PERK、eIF2α和ATF4的表达可以显著被PACE4过表达抑制。结论:PACE4过表达可以抑制ISO诱导的H9c2心肌细胞凋亡,其机制可能与PERK信号通路介导的内质网应激反应有关。     

The double-strand RNA-dependent protein kinase PKR plays a significant role in a sustained ER stress-induced apoptosis

Sustained ER stress leads to apoptosis. However, the exact mechanism still remains to be elucidated. Here, we demonstrate that the double strand RNA-dependent protein kinase (PKR) is involved in the ER stress-mediated signaling pathway. ER stress rapidly activated PKR, inducing the phosphorylation of eIF2alpha, followed by the activation of the ATF4/CHOP pathway. ER-stress-mediated eIF2alpha/ATF4/CHOP signaling and associated cell death was markedly reduced by PKR knockdown. We also found that PKR activation was mediated by PACT, the expression of which was elevated by ER-stress. These results indicate that the ER-stress-mediated eIF2alpha/ATF4/CHOP/cell death pathway is, to some degree, dependent on PACT-mediated PKR activation apart from the PERK pathway.