PRKR-like Endoplasmic Reticulum Kinase Research Articles

Astrocyte PERK and IRE1 Signaling Contributes to Morphine Tolerance and Hyperalgesia through Upregulation of Lipocalin-2 and NLRP3 Inflammasome in the Rodent Spinal Cord.

Endoplasmic reticulum stress plays a crucial role in the pathogenesis of neuroinflammation and chronic pain. This study hypothesized that PRKR-like endoplasmic reticulum kinase (PERK) and inositol-requiring enzyme type 1 (IRE1) regulate lipocalin-2 (LCN2) and Nod-like receptor family pyrin domain containing 3 (NLRP3) expression in astrocytes, thereby contributing to morphine tolerance and hyperalgesia. The study was performed in Sprague-Dawley rats and C57/Bl6 mice of both sexes. The expression of LCN2 and NLRP3 was assessed by Western blotting. The tail-flick, von Frey, and Hargreaves tests were used to evaluate nociceptive behaviors. Chromatin immunoprecipitation was conducted to analyze the binding of activating transcription factor 4 (ATF4) to the promoters of LCN2 and TXNIP. Whole-cell patch-clamp recordings were used to evaluate neuronal excitability. Pharmacologic inhibition of PERK and IRE1 attenuated the development of morphine tolerance and hyperalgesia in male (tail latency on day 7, 8.0 ± 1.13 s in the morphine + GSK2656157 [10 μg] group vs. 5.8 ± 0.65 s in the morphine group; P = 0.04; n = 6 rats/group) and female (tail latency on day 7, 6.0 ± 0.84 s in the morphine + GSK2656157 [10 μg] group vs. 3.1 ± 1.09 s in the morphine group; P = 0.0005; n = 6 rats/group) rats. Activation of PERK and IRE1 upregulated expression of LCN2 and NLRP3 in vivo and in vitro. Chromatin immunoprecipitation analysis showed that ATF4 directly bound to the promoters of the LCN2 and TXNIP. Lipocalin-2 induced neuronal hyperexcitability in the spinal cord and dorsal root ganglia via melanocortin-4 receptor. Astrocyte endoplasmic reticulum stress sensors PERK and IRE1 facilitated morphine tolerance and hyperalgesia through upregulation of LCN2 and NLRP3 in the spinal cord.

PERK/ATF3-Reduced ER Stress on high potassium environment in the suppression of tumor ferroptosis.

Potassium (K+) is a vital intracellular cation. In the human body, it regulates membrane potential, electrical excitation, protein synthesis, and cell death. Recent studies revealed that dying cancer cells release potassium into the tumor microenvironment (TME), thereby influencing cell survival-related events. Several investigations reported that potassium channels and high potassium levels influence apoptosis. Increasing extracellular potassium and inhibiting K+ efflux channels significantly block the apoptotic machinery. However, it is unknown whether a high-potassium environment also affects other types of cell death such as ferroptosis. In the present study, cell counting kit (CCK-8), colony formation ability, and 5-ethynyl-2'-deoxyuridine (EdU) assays demonstrated that a high-potassium environment reverses erastin-induced ferroptosis. RNA sequencing (RNA-Seq) and Kyoto Encyclopedia of Genes and Genomes (KEGG) and gene ontology (GO) analyses indicated that high potassium levels attenuated the unfolded protein response that is characteristic of endoplasmic reticulum (ER) stress. The ER transmembrane proteins PRKR-like ER kinase (PERK), inositol-requiring enzyme 1α (IRE1α), and activating transcription factor 6 (ATF6) are recognized as ER stress sensors. Here, the PERK blocker GSK2606414 significantly rescued ferroptosis. The present work also disclosed that the ER-related gene activating transcription factor 3 (ATF3) played a vital role in regulating ferroptosis in a high-potassium environment. The foregoing results revealed the roles of potassium and the TME in cancer cell ferroptosis and provided a potential clinical therapeutic strategy for cancer.

Exosomal DNAJB11 promotes the development of pancreatic cancer by modulating the EGFR/MAPK pathway

Pancreatic ductal adenocarcinoma (PDAC) is a malignant tumor with invasive and metastatic characteristics and poor prognosis. Intracellular protein homeostasis is associated with invasion and metastasis of pancreatic cancer, but the specific molecular mechanism remains unclear. Our previous studies have revealed that DNAJB11, a key protein in protein homeostasis, is secreted by exosomes in the supernatant of dissociated pancreatic cancer cells with high metastasis. The results from transcriptome sequencing and co-immunoprecipitation (Co-IP)-based liquid chromatography with tandem mass spectrometry (LC–MS/MS) showed that depletion of DNAJB11 levels could increase HSPA5 expression and induce endoplasmic reticulum stress through the PRKR-like endoplasmic reticulum kinase signaling pathway in pancreatic cancer cells. Furthermore, exosomal DNAJB11 promoted cell development of PC cells in vitro and in vivo. In addition, exosomal DNAJB11 could regulate the expression of EGFR and activate the downstream MAPK signaling pathway. Clinical blood samples were collected to evaluate the potential of exosome DNAJB11 as a diagnostic biomarker and therapeutic target for the treatment of pancreatic cancer. This study could provide a new theoretical basis and potential molecular targets for the treatment of pancreatic cancer.

Endoplasmic reticulum stress and the unfolded protein response in skeletal muscle of subjects suffering from peritoneal sepsis

We provide a descriptive characterization of the unfolded protein response (UPR) in skeletal muscle of human patients with peritoneal sepsis and a sepsis model of C57BL/6J mice. Patients undergoing open surgery were included in a cross-sectional study and blood and skeletal muscle samples were taken. Key markers of the UPR and cluster of differentiation 68 (CD68) as surrogate of inflammatory injury were evaluated by real-time PCR and histochemical staining. CD68 mRNA increased with sepsis in skeletal muscle of patients and animals (p < 0.05). Mainly the inositol-requiring enzyme 1α branch of the UPR was upregulated as shown by elevated X-box binding-protein 1 (XBP1u) and its spliced isoform (XBP1s) mRNA (p < 0.05, respectively). Increased expression of Gadd34 indicated activation of PRKR-Like Endoplasmic Reticulum Kinase (PERK) branch of the UPR, and was only observed in mice (p < 0.001) but not human study subjects. Selected cell death signals were upregulated in human and murine muscle, demonstrated by increased bcl-2 associated X protein mRNA and TUNEL staining (p < 0.05). In conclusion we provide a first characterization of the UPR in skeletal muscle in human sepsis.

Metformin alleviates intestinal epithelial barrier damage by inhibiting endoplasmic reticulum stress-induced cell apoptosis in colitis cell model.

To investigate the effect and mechanism of metformin on intestinal epithelial barrier injury in ulcerative colitis. A cell model of colitis was established by co-culture of human colon cancer cell line Caco-2 and human monocyte cell line THP-1. The colitis model cells were treated with metformin at concentration of for Flow cytometry was used to detect Caco-2 cell apoptosis, and Western blotting was used to detect the protein expression of tight junction proteins and endoplasmic reticulum stress-related proteins. After metformin treatment, the apoptosis rate of Caco-2 cells was decreased from (14.22±2.34)% to 0.61)% (=3.119, <0.05), and the expression levels of tight junction protein-1 and claudin-1 increased (=5.172 and 3.546, both <0.05). In addition, the expression levels of endoplasmic reticulum-related proteins glucose regulated protein (GRP) 78, C/EBP homologous protein (CHOP) and caspase-12, as well as the phosphorylation level of PRKR-like endoplasmic reticulum kinase (PERK) and eukaryotic translation initiation factor 2α (eIF2α) decreased (all <0.05). Metformin may alleviate the intestinal epithelial barrier damage in colitis by reducing intestinal epithelial cell apoptosis and increasing the expression of tight junction proteins, which may be associated with the inhibition of endoplasmic reticulum stress-induced apoptotic pathway.

Berberine Reduces Aβ42 Deposition and Tau Hyperphosphorylation via Ameliorating Endoplasmic Reticulum Stress.

Alzheimer’s disease (AD) is tightly related to endoplasmic reticulum stress (ER stress), which aggravates two dominant pathological manifestations of AD: senile plaques and neurofibrillary tangles. Berberine is widely applied in the clinical treatment of many diseases and is reported to have anti-AD effects. In the present study, berberine was shown to ameliorate ER stress and cognitive impairment in APP/PS1 mice. We found ER stress plays a role as a central hub for signal transduction, which was evidenced by the hyperactivation of glycogen synthase kinase 3β (GSK3β) to phosphorylate tau and the activation of PRKR-like endoplasmic reticulum kinase (PERK) subsequently to phosphorylate eukaryotic translation initiation factor-2 α (eIF2α). Also, eIF2α has regulated the expression of beta-site APP cleaving enzyme-1 (BACE1), which cleaves APP into pro-oligomerized amyloid beta 42 (Aβ42), the main component of senile plaques, proven by using siRNA targeting at eIF2α. Mechanically, berberine can reduce GSK3β activity, contributing to the downregulation of tau phosphorylation. Berberine also suppressed Aβ42 production via inhibiting the PERK/eIF2α/BACE1 signaling pathway. Taken together, these findings indicated that berberine had the potential to ameliorate two major pathological manifestations of AD mainly by suppressing ER stress. Our work provided knowledge on the pharmacological intervention of AD and the possible targets for future drug development.

Perspectives on Organelle Interaction, Protein Dysregulation, and Cancer Disease.

In recent decades, compelling evidence has emerged showing that organelles are not static structures but rather form a highly dynamic cellular network and exchange information through membrane contact sites. Although high-throughput techniques facilitate identification of novel contact sites (e.g., organelle-organelle and organelle-vesicle interactions), little is known about their impact on cellular physiology. Moreover, even less is known about how the dysregulation of these structures impacts on cellular function and therefore, disease. Particularly, cancer cells display altered signaling pathways involving several cell organelles; however, the relevance of interorganelle communication in oncogenesis and/or cancer progression remains largely unknown. This review will focus on organelle contacts relevant to cancer pathogenesis. We will highlight specific proteins and protein families residing in these organelle-interfaces that are known to be involved in cancer-related processes. First, we will review the relevance of endoplasmic reticulum (ER)-mitochondria interactions. This section will focus on mitochondria-associated membranes (MAMs) and particularly the tethering proteins at the ER-mitochondria interphase, as well as their role in cancer disease progression. Subsequently, the role of Ca2+ at the ER-mitochondria interphase in cancer disease progression will be discussed. Members of the Bcl-2 protein family, key regulators of cell death, also modulate Ca2+ transport pathways at the ER-mitochondria interphase. Furthermore, we will review the role of ER-mitochondria communication in the regulation of proteostasis, focusing on the ER stress sensor PERK (PRKR-like ER kinase), which exerts dual roles in cancer. Second, we will review the relevance of ER and mitochondria interactions with other organelles. This section will focus on peroxisome and lysosome organelle interactions and their impact on cancer disease progression. In this context, the peroxisome biogenesis factor (PEX) gene family has been linked to cancer. Moreover, the autophagy-lysosome system is emerging as a driving force in the progression of numerous human cancers. Thus, we will summarize our current understanding of the role of each of these organelles and their communication, highlighting how alterations in organelle interfaces participate in cancer development and progression. A better understanding of specific organelle communication sites and their relevant proteins may help to identify potential pharmacological targets for novel therapies in cancer control.

Subtilase cytotoxin induces a novel form of Lipocalin 2, which promotes Shiga-toxigenic Escherichia coli survival

Shiga-toxigenic Escherichia coli (STEC) infection causes severe bloody diarrhea, renal failure, and hemolytic uremic syndrome. Recent studies showed global increases in Locus for Enterocyte Effacement (LEE)-negative STEC infection. Some LEE-negative STEC produce Subtilase cytotoxin (SubAB), which cleaves endoplasmic reticulum (ER) chaperone protein BiP, inducing ER stress and apoptotic cell death. In this study, we report that SubAB induces expression of a novel form of Lipocalin-2 (LCN2), and describe its biological activity and effects on apoptotic cell death. SubAB induced expression of a novel LCN2, which was regulated by PRKR-like endoplasmic reticulum kinase via the C/EBP homologous protein pathway. SubAB-induced novel-sized LCN2 was not secreted into the culture supernatant. Increased intracellular iron level by addition of holo-transferrin or FeCl3 suppressed SubAB-induced PARP cleavage. Normal-sized FLAG-tagged LCN2 suppressed STEC growth, but this effect was not seen in the presence of SubAB- or tunicamycin-induced unglycosylated FLAG-tagged LCN2. Our study demonstrates that SubAB-induced novel-sized LCN2 does not have anti-STEC activity, suggesting that SubAB plays a crucial role in the survival of LEE-negative STEC as well as inducing apoptosis of the host cells.

P0015SIRT2 REGULATES CISPLATIN-INDUCED ENDOPLASMIC RETICULUM STRESS THROUGH HEAT SHOCK FACTOR 1 DEACETYLATION

Abstract Background and Aims Nephrotoxicity is an important cisplatin-induced adverse reaction and restricts the use of cisplatin to treat malignant tumors. Endoplasmic reticulum (ER) stress is caused by the accumulation of misfolded proteins, and is induced by cisplatin in kidneys. SIRT2 nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase is a member of the sirtuin family, but its role in cisplatin-induced ER stress remains unclear. Method To investigate the effect of SIRT2 on cisplatin-induced ER stress using SIRT2 knockout mice and human proximal tubular epithelial cells (HK-2 cells). We treated cisplatin (20 µg/mL) or induced by intraperitoneal injection of cisplatin (20 mg/kg) and evaluated the changes of ER stress and its signal mechanism. Results Cisplatin administration was found to significantly increase the expressions of PRKR-like ER kinase (PERK), phosphorylation of eukaryotic translation initiation factor 2α (eIF2α), and the C/EBP homologous protein (CHOP) and caspase-12 in the kidneys of SIRT2-wild type mice. However, cisplatin-induced increases in the expressions of p-PERK, p-eIF2α, CHOP and, caspase-12 were diminished in kidneys of SIRT2 knockout mice. In vitro, cisplatin significantly increased the expressions of p-PERK, p-eIF2α, CHOP, and caspase-12 in HK-2 cells. When the effect of SIRT2 on cisplatin-induced ER stress was evaluated using SIRT2-siRNA (ON-TARGET plus human SIRT2 siRNA) or the SIRT2 inhibitors, AGK2 and AK1, knockdown or inhibition of SIRT2 significantly attenuated the cisplatin-induced protein expression of p-PERK, p-eIF2α, CHOP, and caspase-12. Immunoprecipitation studies showed SIRT2 bound physically to heat shock factor (HSF)1 and that HSF1 acetylation was significantly increased by cisplatin. In addition, knockdown of SIRT2 increased cisplatin-induced HSF1 acetylation and increased the expression of heat shock protein (HSP)70. Conclusion These observations suggest that suppression of SIRT2 ameliorates cisplatin-induced ER stress by increasing HSF1 acetylation and HSP expression.

Calpain-2 activity promotes aberrant endoplasmic reticulum stress-related apoptosis in hepatocytes.

BACKGROUNDCalpain-2 is a Ca2+-dependent cysteine protease, and high calpain-2 activity can enhance apoptosis mediated by multiple triggers.AIMTo investigate whether calpain-2 can modulate aberrant endoplasmic reticulum (ER) stress-related apoptosis in rat hepatocyte BRL-3A cells.METHODSBRL-3A cells were treated with varying doses of dithiothreitol (DTT), and their viability and apoptosis were quantified by 3-[4, 5-dimethyl-2-thiazolyl]-2, 5-diphenyl-2-H-tetrazolium bromide and flow cytometry. The expression of ER stress- and apoptosis-related proteins was detected by Western blot analysis. The protease activity of calpain-2 was determined using a fluorescent substrate, N-succinyl-Leu-Leu-Val-Tyr-AMC. Intracellular Ca2+ content, and ER and calpain-2 co-localization were characterized by fluorescent microscopy. The impact of calpain-2 silencing by specific small interfering RNA on caspase-12 activation and apoptosis of BRL-3A cells was quantified.RESULTSDTT exhibited dose-dependent cytotoxicity against BRL-3A cells and treatment with 2 mmol/L DTT triggered BRL-3A cell apoptosis. DTT treatment significantly upregulated 78 kDa glucose-regulated protein, activating transcription factor 4, C/EBP-homologous protein expression by >2-fold, and enhanced PRKR-like ER kinase phosphorylation, caspase-12 and caspase-3 cleavage in BRL-3A cells in a trend of time-dependence. DTT treatment also significantly increased intracellular Ca2+ content, calpain-2 expression, and activity by >2-fold in BRL-3A cells. Furthermore, immunofluorescence revealed that DTT treatment promoted the ER accumulation of calpain-2. Moreover, calpain-2 silencing to decrease calpain-2 expression by 85% significantly mitigated DTT-enhanced calpain-2 expression, caspase-12 cleavage, and apoptosis in BRL-3A cells.CONCLUSIONThe data indicated that Ca2+-dependent calpain-2 activity promoted the aberrant ER stress-related apoptosis of rat hepatocytes by activating caspase-12 in the ER.

C6-ceramide induces salivary adenoid cystic carcinoma cell apoptosis via IP3R-activated UPR and UPR-independent pathways

C6-ceramide is an exogenous short-chain ceramide which can induce apoptosis of multiple cancer cells. Salivary adenoid cystic carcinoma (SACC) is a common salivary gland cancer, which possesses of high rate of local recurrence and distant metastasis. The mechanism of ceramide-induced SACC-83 and SACC-LM cell apoptosis has not been revealed. In our study, gene expression microarray was used to discover that the unfolded protein response (UPR) pathway, especially PRKR-like endoplasmic reticulum kinase (PERK) pathway, was the major activated pathway after treatment of c6-ceramide. D1ER, an endoplasmic-reticulum-targeted Ca2+ indicator, was used to measure Ca2+ release from endoplasmic reticulum (ER) dynamically. We found that inositol 1,4,5-trisphosphate receptor 3 (IP3R3) was activated, leading to Ca2+ release from ER, soon after c6-ceramide treatment. IP3R3 silencing could block UPR, although it could not prevent SACC-83 and SACC-LM cells from apoptosis. Moreover, we found that C/EBP-homologous protein could upregulate in a UPR-independent way. Mitochondria outer membrane permeabilization might play an important role in inducing SACC cell apoptosis.

Dienogest regulates apoptosis, proliferation, and invasiveness of endometriotic cyst stromal cells via endoplasmic reticulum stress induction.

Dienogest, a specific progesterone receptor agonist, is used in the treatment of endometriosis. However, it is still unclear as to the mechanisms of therapeutic effects on endometriosis. Our recent study showed that endometriosis may be the result of aberrant endoplasmic reticulum (ER) stress induction due to progesterone resistance. This finding suggests that the regulation of ER stress induction may play a key role in treatment of endometriosis. Therefore, the anti-endometriotic effects of dienogest may be mediated by regulation of ER stress. To test this hypothesis, we elucidate whether dienogest affects endometriotic stromal cell apoptosis, proliferation and invasiveness by modulating ER stress-induced CCAAT/enhancer-binding protein homologous protein (CHOP) expression. Specifically, PRKR-like ER kinase (PERK)/eukaryotic initiation factor 2α (eIF2α)/activating transcription factor 4 (ATF4), inositol-requiring kinase 1 (IRE1)/TNF receptor-associated factor 2 (TRAF2)/apoptosis signal-regulating kinase 1 (ASK1)/c-Jun N-terminal kinase (JNK) signaling, and downstream CHOP were evaluated to determine the involved ER stress-mediated regulation mechanism of CHOP expression. Our results show that progesterone treatment did not have any significant effects on ER stress, apoptosis, proliferation, and invasion in estrogen-treated endometriotic cyst stromal cells (ECSCs). However, dienogest treatment upregulated the induction of ER stress. It also led to increased apoptosis, and decreased proliferation and invasiveness. These dienogest-induced changes in apoptosis, proliferation and invasiveness were reversed by the ER stress inhibitor salubrinal. Furthermore, dienogest-induced ER stress increased CHOP expression through activation of both PERK/elf2α/ATF4 and IRE1/TRAF2/ASK1/JNK signaling. This upregulation was blocked by transfection with PERK and IRE1 siRNA, which decreased apoptosis and increased the proliferation and invasiveness of dienogest-treated ECSCs. Taken together, our findings indicate that dienogest enhances ER stress induction in endometriotic stromal cells, which affects apoptosis, proliferation and invasiveness via CHOP upregulation.

The Endoplasmic Reticulum and Calcium Homeostasis in Pancreatic Beta Cells.

The endoplasmic reticulum (ER) mediates the first steps of protein assembly within the secretory pathway and is the site where protein folding and quality control are initiated. The storage and release of Ca2+ are critical physiological functions of the ER. Disrupted ER homeostasis activates the unfolded protein response (UPR), a pathway which attempts to restore cellular equilibrium in the face of ER stress. Unremitting ER stress, and insufficient compensation for it results in beta-cell apoptosis, a process that has been linked to both type 1 diabetes (T1D) and type 2 diabetes (T2D). Both types are characterized by progressive beta-cell failure and a loss of beta-cell mass, although the underlying causes are different. The reduction of mass occurs secondary to apoptosis in the case of T2D, while beta cells undergo autoimmune destruction in T1D. In this review, we examine recent findings that link the UPR pathway and ER Ca2+ to beta cell dysfunction. We also discuss how UPR activation in beta cells favors cell survival versus apoptosis and death, and how ER protein chaperones are involved in regulating ER Ca2+ levels. Abbreviations: BiP, Binding immunoglobulin Protein ER; endoplasmic reticulum; ERAD, ER-associated protein degradation; IFN, interferon; IL, interleukin; JNK, c-Jun N-terminal kinase; KHE, proton-K+ exchanger; MODY, maturity-onset diabetes of young; PERK, PRKR-like ER kinase; SERCA, Sarco/Endoplasmic Reticulum Ca2+-ATPases; T1D, type 1 diabetes; T2D, type 2 diabetes; TNF, tumor necrosis factor; UPR, unfolded protein response; WRS, Wolcott-Rallison syndrome.

A guide to assessing endoplasmic reticulum homeostasis and stress in mammalian systems.

The endoplasmic reticulum (ER) is a multifunctional organelle that constitutes the entry into the secretory pathway. The ER contributes to the maintenance of cellular calcium homeostasis, lipid synthesis and productive secretory, and transmembrane protein folding. Physiological, chemical, and pathological factors that compromise ER homeostasis lead to endoplasmic reticulum stress (ER stress). To cope with this situation, cells activate an adaptive signaling pathway termed the unfolded protein response (UPR) that aims at restoring ER homeostasis. The UPR is transduced through post-translational, translational, post-transcriptional, and transcriptional mechanisms initiated by three ER-resident sensors, inositol-requiring protein 1α, activating transcription factor 6α, and PRKR-like endoplasmic reticulum kinase. Determining the in and out of ER homeostasis control and UPR activation still represents a challenge for the community. Hence, standardized criteria and methodologies need to be proposed for monitoring ER homeostasis and ER stress in different model systems. Here, we summarize the pathways that are activated during ER stress and provide approaches aimed at assess ER homeostasis and stress invitro and invivo mammalian systems that can be used by researchers to plan and interpret experiments. We recommend the use of multiple assays to verify ER stress because no individual assay is guaranteed to be the most appropriate one.

Suberoylanilide hydroxamic acid upregulates histone acetylation and activates endoplasmic reticulum stress to induce apoptosis in HepG2 liver cancer cells.

Suberoylanilide hydroxamic acid (SAHA) is a histone deacetylase inhibitor that has demonstrated clinical activity against various solid tumors. The aim of the present study was to explore the effects of SAHA on the apoptosis of HepG2 liver cancer cells, as well as the potential mechanisms involved in histone acetylation and endoplasmic reticulum (ER) stress. HepG2 cells were treated with various doses of SAHA (0, 1, 6 and 12 µM), and apoptosis was measured by flow cytometry. The levels of ER stress-associated molecules, including 78 kDa glucose-regulated protein (GRP78), PRKR-like endoplasmic reticulum kinase (PERK), phosphorylated (p)-PERK, activating transcription factor 4 (ATF4) and C/EBP-homologous protein (CHOP), were quantitated by western blot analysis and reverse transcription-quantitative PCR assay. The expression levels of acetylated histone H4 (acH4, acH4 lysine (K)5 and acH4K12) were detected by western blot analysis. The effects of SAHA on the acetylation of H4 in the promoter regions of GRP78, ATF4 and CHOP were evaluated by chromatin immunoprecipitation assays. Following treatment with higher doses of SAHA (6 and 12 µM) for 48 h, the proliferation of HepG2 cells was significantly suppressed. SAHA induced dose-dependent apoptosis and increased both protein and mRNA expression levels of GRP78, ATF4 and CHOP in HepG2 cells. The protein expression of PERK was markedly decreased by treatment with SAHA, whereas the p-PERK expression level was notably increased, which resulted in increased p-PERK/PERK ratio. Furthermore, the acetylation levels of H4 in the promoter regions of GRP78, ATF4 and CHOP were significantly increased in HepG2 cells exposed to 6 µM SAHA for 36 h. Thus, SAHA induces apoptosis in HepG2 cells by activating the ER stress-mediated apoptotic signaling pathway, at least partially by enhancing the acetylation of histone H4 on the promoter regions of ER-stress associated genes, including GRP78, ATF4 and CHOP.

ER stress signaling has an activating transcription factor 6α (ATF6)-dependent “off-switch”

In response to an accumulation of unfolded proteins in the endoplasmic reticulum (ER) lumen, three ER transmembrane signaling proteins, inositol-requiring enzyme 1 (IRE1), PRKR-like ER kinase (PERK), and activating transcription factor 6α (ATF6α), are activated. These proteins initiate a signaling and transcriptional network termed the unfolded protein response (UPR), which re-establishes cellular proteostasis. When this restoration fails, however, cells undergo apoptosis. To investigate cross-talk between these different UPR enzymes, here we developed a high-content live cell screening platform to image fluorescent UPR-reporter cell lines derived from human SH-SY5Y neuroblastoma cells in which different ER stress signaling proteins were silenced through lentivirus-delivered shRNA constructs. We observed that loss of ATF6 expression results in uncontrolled IRE1-reporter activity and increases X box-binding protein 1 (XBP1) splicing. Transient increases in both IRE1 mRNA and IRE1 protein levels were observed in response to ER stress, suggesting that IRE1 up-regulation is a general feature of ER stress signaling and was further increased in cells lacking ATF6 expression. Moreover, overexpression of the transcriptionally active N-terminal domain of ATF6 reversed the increases in IRE1 levels. Furthermore, inhibition of IRE1 kinase activity or of downstream JNK activity prevented an increase in IRE1 levels during ER stress, suggesting that IRE1 transcription is regulated through a positive feed-forward loop. Collectively, our results indicate that from the moment of activation, IRE1 signaling during ER stress has an ATF6-dependent "off-switch."

Apoptosis-Associated Speck-Like Protein Containing a CARD Deletion Ameliorates Unilateral Ureteral Obstruction Induced Renal Fibrosis and Endoplasmic Reticulum Stress in Mice.

Inflammation might be one of the essential underlying mechanisms of renal fibrosis, which is considered a key pathological feature of end-stage renal disease and is closely associated with proteinuria and decreased renal function. Apoptosis-associated speck-like protein containing a CARD (ASC), identified as the central structure of inflammasome, is involved in the progression of interstitial fibrosis; however, its signal transduction pathways remain unclear. In the present study, we performed unilateral ureter obstruction (UUO) in both wild-type and ASC deletion mice to determine the contribution of ASC to renal fibrosis. Compared with control groups, UUO significantly induced renal fibrosis and collagen deposition, as evidenced by photomicrographs. ASC deletion attenuated renal injury, reduced cell infiltration and the release of inflammatory cytokines, protected against apoptosis, and downregulated the PRKR-like endoplasmic reticulum kinase (PERK) pathway of endoplasmic reticulum (ER) stress. Our data identify a novel role of ASC in the regulation of renal fibrosis and ER stress after UUO, strongly indicating that ASC could serve as an attractive target in the treatment of chronic kidney disease.

Chrysin induces cell growth arrest, apoptosis, and ER stress and inhibits the activation of STAT3 through the generation of ROS in bladder cancer cells.

Chrysin is a natural flavone that has various biological activities, including antitumor effects. However, the effect of chrysin on bladder cancer cells remains elusive. The present study investigated the effects of chrysin on bladder cancer cells and its underlying mechanisms. The results demonstrated that chrysin induced apoptosis via the intrinsic pathway, as evidenced by activation of caspase-9 and caspase-3, however not caspase-8. In addition, chrysin reduced the expression of anti-apoptotic B cell lymphoma (Bcl) proteins including Bcl-2, Mcl-1, Bcl-xl, and promoted the protein expression of pro-apoptotic Bcl-2 associated X, apoptosis regulator. Chrysin also induced endoplasmic reticulum stress via activation of the unfolded protein response of PRKR-like endoplasmic reticulum kinase, eIF2α and activating transcription factor 4 in bladder cancer cells. Additionally, chrysin inhibited the signal transducer and activator of transcription 3 pathway. Furthermore, the generation of reactive oxygen species (ROS) was detected following treatment with chrysin. The ROS scavenger N-acetylcysteine inhibited the antitumor effect of chrysin. Collectively, these results indicate chrysin may act as a promising therapeutic candidate for targeting bladder cancer.

The luminal domain of the ER stress sensor protein PERK binds misfolded proteins and thereby triggers PERK oligomerization

PRKR-like endoplasmic reticulum kinase (PERK) is one of the major sensor proteins that detect protein folding imbalances during endoplasmic reticulum (ER) stress. However, it remains unclear how ER stress activates PERK to initiate a downstream unfolded protein response (UPR). Here, we found that PERK's luminal domain can recognize and selectively interact with misfolded proteins but not with native proteins. Screening a phage-display library, we identified a peptide substrate, P16, of the PERK luminal domain and confirmed that P16 efficiently competes with misfolded proteins for binding this domain. To unravel the mechanism by which the PERK luminal domain interacts with misfolded proteins, we determined the crystal structure of the bovine PERK luminal domain complexed with P16 to 2.8-Å resolution. The structure revealed that PERK's luminal domain binds the peptide through a conserved hydrophobic groove. Substitutions within hydrophobic regions of the PERK luminal domain abolished the binding between PERK and misfolded proteins. We also noted that peptide binding results in major conformational changes in the PERK luminal domain that may favor PERK oligomerization. The structure of the PERK luminal domain-P16 complex suggested stacking of the luminal domain that leads to PERK oligomerization and activation via autophosphorylation after ligand binding. Collectively, our structural and biochemical results strongly support a ligand-driven model in which the PERK luminal domain interacts directly with misfolded proteins to induce PERK oligomerization and activation, resulting in ER stress signaling and the UPR.

Chrysin induces death of prostate cancer cells by inducing ROS and ER stress.

Chrysin is a natural flavone found in numerous plant extracts, honey, and propolis that has multiple biological activities including anti-cancer effects. Understanding of biological mechanisms mediated in response to chrysin in cancerous cells may provide novel insight into chemotherapeutic approaches with reduced side effects in cancers. In the present study, we investigated functional roles of chrysin in progression of prostate cancer cells using DU145 and PC-3 cell lines. The results showed that chrysin induced apoptosis of cells evidenced by DNA fragmentation and increasing the population of both DU145 and PC-3 cells in the sub-G1 phase of the cell cycle. In addition, chrysin reduced expression of proliferating cell nuclear antigen in the prostate cancer cell lines compared to untreated prostate cancer cells. Moreover, chrysin induced loss of mitochondria membrane potential (MMP), while increasing production of reactive oxygen species (ROS) and lipid peroxidation in a dose-dependent manner. Also, it induced endoplasmic reticulum (ER) stress through activation of unfolded protein response (UPR) proteins including PRKR-like ER kinase (PERK), eukaryotic translation initiation factor 2α (eIF2α), and 78 kDa glucose-regulated protein (GRP78) in DU145 and PC-3 cells. The chrysin-mediated intracellular signaling pathways suppressed phosphoinositide 3-kinase (PI3K) and the abundance of AKT, P70S6K, S6, and P90RSK proteins, but stimulated mitogen-activated protein kinases (MAPK) and activation of ERK1/2 and P38 proteins in the prostate cancer cells. Collectively, these results indicate that chrysin initiates cell death through induction of mitochondrial-mediated apoptosis and ER stress, and regulation of signaling pathways responsible for proliferation of prostate cancer cells.