p62/SQSTM1 is required for the protection against endoplasmic reticulum stress-induced apoptotic cell death

Endoplasmic reticulum (ER) stress is triggered by various cellular stresses that disturb protein folding or calcium homeostasis in the ER. To cope with these stresses, ER stress activates the unfolded protein response (UPR) pathway, but unresolved ER stress induces reactive oxygen species (ROS) accumulation leading to apoptotic cell death. However, the mechanisms that underlie protection from ER stress-induced cell death are not clearly defined. The nuclear factor erythroid 2-related factor 2 (Nrf2)-Kelch-like ECH-associated protein 1 (Keap1) pathway plays a crucial role in the protection of cells against ROS-mediated oxidative damage. Keap1 acts as a negative regulator of Nrf2 activation. In this study, we investigated the role of the Nrf2-Keap1 pathway in protection from ER stress-induced cell death using tunicamycin (TM) as an ER stress inducer. We found that Nrf2 is an essential protein for the prevention from TM-induced apoptotic cell death and its activation is driven by autophagic Keap1 degradation. Furthermore, ablation of p62, an adapter protein in the autophagy process, attenuates the Keap1 degradation and Nrf2 activation that was induced by TM treatment, and thereby increases susceptibility to apoptotic cell death. Conversely, reinforcement of p62 alleviated TM-induced cell death in p62-deficient cells. Taken together, these results demonstrate that p62 plays an important role in protecting cells from TM-induced cell death through Nrf2 activation.     

PERK-dependent activation of Nrf2 contributes to redox homeostasis and cell survival following endoplasmic reticulum stress

The accumulation of unfolded proteins elicits a cellular response that triggers both pro-survival and pro-apoptotic signaling events. PERK-dependent activation of NF-E2-related factor-2 (Nrf2) is critical for survival signaling during this response; however, the mechanism whereby Nrf2 confers a protective advantage to stressed cells remains to be defined. We now demonstrate that Nrf2 activation contributes to the maintenance of glutathione levels, which in turn functions as a buffer for the accumulation of reactive oxygen species during the unfolded protein response. The deleterious effects of Nrf2 or PERK deficiencies could be attenuated by the restoration of cellular glutathione levels or Nrf2 activity. In addition, the inhibition of reactive oxygen species production attenuated apoptotic induction following endoplasmic reticulum stress. Our data suggest that perturbations in cellular redox status sensitize cells to the harmful effects of endoplasmic reticulum stress, but that other factors are essential for apoptotic commitment.     

A Highlights from MBoC Selection: Translational and posttranslational regulation of XIAP by eIF2α and ATF4 promotes ER stress–induced cell death during the unfolded protein response

Endoplasmic reticulum (ER) protein misfolding activates the unfolded protein response (UPR) to help cells cope with ER stress. If ER homeostasis is not restored, UPR promotes cell death. The mechanisms of UPR-mediated cell death are poorly understood. The PKR-like endoplasmic reticulum kinase (PERK) arm of the UPR is implicated in ER stress–induced cell death, in part through up-regulation of proapoptotic CCAAT/enhancer binding protein homologous protein (CHOP). Chop^−/− cells are partially resistant to ER stress–induced cell death, and CHOP overexpression alone does not induce cell death. These findings suggest that additional mechanisms regulate cell death downstream of PERK. Here we find dramatic suppression of antiapoptosis XIAP proteins in response to chronic ER stress. We find that PERK down-regulates XIAP synthesis through eIF2α and promotes XIAP degradation through ATF4. Of interest, PERK''s down-regulation of XIAP occurs independently of CHOP activity. Loss of XIAP leads to increased cell death, whereas XIAP overexpression significantly enhances resistance to ER stress–induced cell death, even in the absence of CHOP. Our findings define a novel signaling circuit between PERK and XIAP that operates in parallel with PERK to CHOP induction to influence cell survival during ER stress. We propose a “two-hit” model of ER stress–induced cell death involving concomitant CHOP up-regulation and XIAP down-regulation both induced by PERK.     

Activation of the UPR Protects against Cigarette Smoke-induced RPE Apoptosis through Up-Regulation of Nrf2

Recent studies have revealed a role of endoplasmic reticulum (ER) stress-induced unfolded protein response (UPR) in the regulation of RPE cell activity and survival. Herein, we examined the mechanisms by which the UPR modulates apoptotic signaling in human RPE cells challenged with cigarette smoking extract (CSE). Our results show that CSE exposure induced a dose- and time-dependent increase in ER stress markers, enhanced reactive oxygen species (ROS), mitochondrial fragmentation, and apoptosis of RPE cells. These changes were prevented by the anti-oxidant NAC or chemical chaperone TMAO, suggesting a close interaction between oxidative and ER stress in CSE-induced apoptosis. To decipher the role of the UPR, overexpression or down-regulation of XBP1 and CHOP genes was manipulated by adenovirus or siRNA. Overexpressing XBP1 protected against CSE-induced apoptosis by reducing CHOP, p-p38, and caspase-3 activation. In contrast, XBP1 knockdown sensitized the cells to CSE-induced apoptosis, which is likely through a CHOP-independent pathway. Surprisingly, knockdown of CHOP reduced p-eIF2α and Nrf2 resulting in a marked increase in caspase-3 activation and apoptosis. Furthermore, Nrf2 inhibition increased ER stress and exacerbated cell apoptosis, while Nrf2 overexpression reduced CHOP and protected RPE cells. Our data suggest that although CHOP may function as a pro-apoptotic gene during ER stress, it is also required for Nrf2 up-regulation and RPE cell survival. In addition, enhancing Nrf2 and XBP1 activity may help reduce oxidative and ER stress and protect RPE cells from cigarette smoke-induced damage.     

Methylglyoxal induces endoplasmic reticulum stress and DNA demethylation in the Keap1 promoter of human lens epithelial cells and age-related cataracts

Age-related cataracts are a leading cause of blindness. Previously, we have demonstrated the association of the unfolded protein response with various cataractogenic stressors. However, DNA methylation alterations leading to suppression of lenticular antioxidant protection remains unclear. Here, we report the methylglyoxal-mediated sequential events responsible for Keap1 promoter DNA demethylation in human lens epithelial cells, because Keap1 is a negative regulatory protein that regulates the Nrf2 antioxidant protein. Methylglyoxal induces endoplasmic reticulum stress and activates the unfolded protein response leading to overproduction of reactive oxygen species before human lens epithelial cell death. Methylglyoxal also suppresses Nrf2 and DNA methyltransferases but activates the DNA demethylation pathway enzyme TET1. Bisulfite genomic DNA sequencing confirms the methylglyoxal-mediated Keap1 promoter DNA demethylation leading to overexpression of Keap1 mRNA and protein. Similarly, bisulfite genomic DNA sequencing shows that human clear lenses (n = 15) slowly lose 5-methylcytosine in the Keap1 promoter throughout life, at a rate of 1% per year. By contrast, diabetic cataractous lenses (n = 21) lose an average of 90% of the 5-methylcytosine regardless of age. Overexpressed Keap1 protein is responsible for decreasing Nrf2 by proteasomal degradation, thereby suppressing Nrf2-dependent stress protection. This study demonstrates for the first time the associations of unfolded protein response activation, Nrf2-dependent antioxidant system failure, and loss of Keap1 promoter methylation because of altered active and passive DNA demethylation pathway enzymes in human lens epithelial cells by methylglyoxal. As an outcome, the cellular redox balance is altered toward lens oxidation and cataract formation.     

Dynamic interaction of BiP and ER stress transducers in the unfolded-protein response

PERK and IRE1 are type-I transmembrane protein kinases that reside in the endoplasmic reticulum (ER) and transmit stress signals in response to perturbation of protein folding. Here we show that the lumenal domains of these two proteins are functionally interchangeable in mediating an ER stress response and that, in unstressed cells, both lumenal domains form a stable complex with the ER chaperone BiP. Perturbation of protein folding promotes reversible dissociation of BiP from the lumenal domains of PERK and IRE1. Loss of BiP correlates with the formation of high-molecular-mass complexes of activated PERK or IRE1, and overexpression of BiP attenuates their activation. These findings are consistent with a model in which BiP represses signalling through PERK and IRE1 and protein misfolding relieves this repression by effecting the release of BiP from the PERK and IRE1 lumenal domains.     

Ligand-independent dimerization activates the stress response kinases IRE1 and PERK in the lumen of the endoplasmic reticulum

IRE1 and PERK are type I transmembrane serine/threonine protein kinases that are activated by unfolded proteins in the endoplasmic reticulum (ER) to signal adaptive responses. IRE1 is present in all eukaryotic cells and signals the unfolded protein response through its kinase and endoribonuclease activities. PERK signals phosphorylation of a translation initiation factor to inhibit protein synthesis in higher eukaryotic cells but is absent in the Saccharomyces cerevisiae genome. The amino acid sequences of the amino-terminal ER luminal domains (NLDs) from IRE1 and PERK display limited homology and have diverged among species. In this study, we have demonstrated that the NLD of yeast Ire1p is required for signaling. However, the NLDs from human IRE1alpha and murine IRE1beta and the Caenorhabditis elegans IRE1 and PERK function as replacements for the S. cerevisiae Ire1p-NLD to signal the unfolded protein response. Replacement of the Ire1p-NLD with a functional leucine zipper dimerization motif yielded a constitutively active kinase that surprisingly was further activated by ER stress. These results demonstrate that ER stress-induced dimerization of the NLD is sufficient for IRE1 and PERK activation and is conserved through evolution. We propose that ligand-independent activation of IRE1 and PERK permits homodimerization upon accumulation of unfolded proteins in the lumen of the ER.     

氧化和化学应激的防御性转导通路——Nrf2/ARE

Nrf2/ARE是近年新发现的机体抵抗内外界氧化和化学等刺激的防御性转导通路．生理条件下,NF-E2相关因子2(Nrf2,NF-E2-related factor 2)在细胞质中与Keap1结合处于非活性、易降解的状态.在内外界自由基和化学物质刺激时,Keap1的构象改变或者Nrf2直接被磷酸化,导致Nrf2与Keap1解离而活化．活化的Nrf2进入细胞核,与抗氧化反应元件(ARE)结合,启动ARE下游的Ⅱ相解毒酶、抗氧化蛋白、蛋白酶体/分子伴侣等基因转录和表达以抵抗内外界的有害刺激．MAPK、PI3K/AKT、PKC等信号通路分子广泛参与了Nrf2的活化和核转位过程,但是具体何种通路被激动、何种通路发挥主导作用,取决于刺激物种类、刺激方式和细胞类型．本文就Nrf2分子结构、Nrf2活化机制、Nrf2/ARE调控的下游基因、与Nrf2相关的信号通路分子以及其在肿瘤、炎症、衰老等应用领域的最新进展进行综述．     

Selenite cataracts: Activation of endoplasmic reticulum stress and loss of Nrf2/Keap1-dependent stress protection

Cataract-induced by sodium selenite in suckling rats is one of the suitable animal models to study the basic mechanism of human cataract formation. The aim of this present investigation is to study the endoplasmic reticulum (ER) stress-mediated activation of unfolded protein response (UPR), overproduction of reactive oxygen species (ROS), and suppression of Nrf2/Keap1-dependent antioxidant protection through endoplasmic reticulum-associated degradation (ERAD) pathway and Keap1 promoter DNA demethylation in human lens epithelial cells (HLECs) treated with sodium selenite. Lenses enucleated from sodium selenite injected rats generated overproduction of ROS in lens epithelial cells and newly formed lens fiber cells resulting in massive lens epithelial cells death after 1–5 days. All these lenses developed nuclear cataracts after 4–5 days. Sodium selenite treated HLECs induced ER stress and activated the UPR leading to release of Ca^2 + from ER, ROS overproduction and finally HLECs death. Sodium selenite also activated the mRNA expressions of passive DNA demethylation pathway enzymes such as Dnmt1, Dnmt3a, and Dnmt3b, and active DNA demethylation pathway enzyme, Tet1 leading to DNA demethylation in the Keap1 promoter of HLECs. This demethylated Keap1 promoter results in overexpression of Keap1 mRNA and protein. Overexpression Keap1 protein suppresses the Nrf2 protein through ERAD leading to suppression of Nrf2/Keap1 dependent antioxidant protection in the HLECs treated with sodium selenite. As an outcome, the cellular redox status is altered towards lens oxidation and results in cataract formation.     

Activation of PERK in ET-1- and thrombin-induced pulmonary fibroblast differentiation: Inhibitory effects of curcumin

In the present study, we investigated the role of PKR-like endoplasmic reticular kinase (PERK), an endoplasmic reticulum (ER) stress kinase, in endothelin 1 (ET-1)- and thrombin-induced pulmonary fibrosis (PF), and the preventive effects of curcumin (CUR). Using the human embryonic WI-38 lung fibroblast cell line, ET-1 and thrombin induced the expression of ER stress-related proteins (CCAAT-enhancer-binding protein homologous protein, PERK, and binding immunoglobulin protein), a profibrogenic factor (cellular communication network factor 2 [CCN2]), and differentiation markers including α-smooth muscle actin (α-SMA), collagen I (Col I), and Col IV. Knockdown of PERK expression via small interfering RNA (siRNA) significantly reduced the increases in CCN2, α-SMA, Col I, and Col IV proteins in WI-38 cells according to western blot analysis and immunohistochemistry (IHC). Activation of c-Jun N-terminal kinase (JNK) was observed in ET-1- and thrombin-treated WI-38 cells, and the addition of a JNK inhibitor (SP) suppressed the induction of the indicated proteins by ET-1 and thrombin. Thapsigargin (TG), an ER stress inducer, elevated expressions of PERK and ER stress-related proteins with increased differentiation of WI-38 cells. Knockdown of PERK by siRNA or the PERK inhibitor glycogen synthesis kinase reduced expressions of the differentiation markers, α-SMA and Col IV, in WI-38 cells. CUR concentration-dependently inhibited ET-1- or thrombin-induced CCN2, α-SMA, and vimentin proteins with decreased levels of phosphorylated mitogen-activated protein kinase and PERK in WI-38 cells. An in vivo bleomycin-induced PF study showed that an intraperitoneal injection of CUR (30 mg/kg) reduced expressions of α-SMA, CCN2, Col IV, and vimentin in lung tissues via IHC staining using specific antibodies. This study is the first to demonstrate that PERK activation contributes to pulmonary fibroblast differentiation elicited by ET-1 or thrombin, and the inhibitory activity of CUR against PF is demonstrated herein.