Search

The protein kinase Gcn2 is a central transducer of nutritional stress signaling important for stress adaptation by normal cells and the survival of cancer cells. In response to nutrient deprivation, Gcn2 phosphorylates eIF2α, thereby repressing general translation while enhancing translation of specific mRNAs with upstream ORFs (uORFs) situated in their 5′-leader regions. Here we performed genome-wide measurements of mRNA translation during histidine starvation in fission yeast Schizosaccharomyces pombe. Polysome analyses were combined with microarray measurements to identify gene transcripts whose translation was up-regulated in response to the stress in a Gcn2-dependent manner. We determined that translation is reprogrammed to enhance RNA metabolism and chromatin regulation and repress ribosome synthesis. Interestingly, translation of intron-containing mRNAs was up-regulated. The products of the regulated genes include additional eIF2α kinase Hri2 amplifying the stress signaling and Gcn5 histone acetyl transferase and transcription factors, together altering genome-wide transcription. Unique dipeptide-coding uORFs and nucleotide motifs, such as ‘5′-UGA(C/G)GG-3′, are found in 5′ leader regions of regulated genes and shown to be responsible for translational control.

The endogenous non-coding microRNA (miRNA) let-7b-5p is highly expressed in the blood of patients with acute pulmonary embolism (PE). However, the mechanism underlying the involvement of let-7b-5p in acute PE remains unclear. To address this, we investigated the role of let-7b-5p in acute PE in both in vitro and in vivo experimental models. The results showed that let-7b-5p upregulated the expression of stress-associated endoplasmic reticulum protein 1 (SERP1) at the post-transcriptional level. SERP1 activation leads to modulation of its chaperone protein SEC61B in the response of endoplasmic reticulum (ER) stress. Furthermore, our data show that the unfolded protein response was triggered and activation of unfolded proteins GRP78, PERK, RNF121, and CHOP occurred through the PERK-CHOP pathway, resulting in an inflammatory response and apoptosis of lung epithelial cells. These characteristics were promoted by the in vitro expression of a let-7b-5p mimic; conversely, transfection with a let-7b-5p inhibitor decreased the response of ER stress in acute PE. The results from this study thus provide evidence that let-7b-5p promotes protein processing during ER stress response by upregulating SERP1 expression, ultimately resulting in an inflammatory response and apoptosis of lung cells, cumulatively playing a critical role in the pathogenesis of acute PE.

Endoplasmic reticulum (ER) stress is associated with several aging-related diseases; however, the mechanism underlying age-related deterioration of oocyte quality is unclear. Here, we used post-ovulatory, in vivo aged mouse oocytes as a model. Super-ovulated oocytes harvested from the oviduct at 14 h and 20 h post-hCG injection were designated as ‘fresh’ and ‘aged’, respectively. Embryo development following IVF was compared between fresh, aged and ER stress-induced oocytes. Expression of the ER stress marker GRP78 was examined at each stage. To evaluate the effect of salubrinal, an ER stress suppressor, on embryo development following IVF, expression levels of GRP78 and phospho-eukaryotic initiation factor 2 alpha were compared between aged and salubrinal-treated aged oocytes. Embryo transfer of salubrinal-treated aged oocytes was performed to examine the safety of salubrinal. Similar to aged oocytes, ER stress-induced oocytes showed lower fertilization rates and poor embryo development. Following IVF, expression of GRP78 decreased with embryo development. GRP78 expression was significantly higher in aged oocytes than in fresh oocytes. Salubrinal lowered GRP78 levels and improved embryo development. No adverse effect of salubrinal treatment was found on the birth weight of pups or on organogenesis in mice. The limitation of this study was that protein kinase-like ER kinase was the only ER stress pathway examined; the role of IRE1 and ATF6 pathways was not considered. Nevertheless, salubrinal can significantly improve embryo development in in vivo aged oocytes undergoing ER stress. Hence, regulation of ER stress might represent a promising therapeutic strategy to overcome poor oocyte quality.

Transcriptional programs regulated by the NF-κB family are essential for the inflammatory response as well as for innate and adaptive immunity. NF-κB activation occurs via two major signaling pathways: the canonical and the noncanonical. The canonical NF-κB pathway responds to diverse immune stimulations and leads to rapid but transient activation. As a member of the canonical NF-κB family, p65 is thought to be a key regulator of viral infection. Because of the embryonic lethality of p65-null mice, the physiological role of p65 in the antiviral immune response is still unclear. In this study, we generated p65-null zebrafish, which were viable and indistinguishable from their wildtype (WT) siblings under normal conditions. However, p65-null zebrafish were more sensitive to spring viremia of carp virus infection than their WT siblings. Further assays indicated that proinflammatory and antiviral genes, including IFN, were downregulated in p65-null zebrafish after spring viremia of carp virus infection compared with their WT siblings. Our results thus suggested that p65 is required for the antiviral response, activating not only proinflammatory genes but also antiviral genes (including IFN).

It is well accepted that cancers co-opt the microenvironment for their growth. However, the molecular mechanisms that underlie cancer-microenvironment interactions are still poorly defined. Here, we show that Rho-associated kinase (ROCK) in the mammary tumour epithelium selectively actuates protein-kinase-R-like endoplasmic reticulum kinase (PERK), causing the recruitment and persistent education of tumour-promoting cancer-associated fibroblasts (CAFs), which are part of the cancer microenvironment. An analysis of tumours from patients and mice reveals that cysteine-rich with EGF-like domains 2 (CRELD2) is the paracrine factor that underlies PERK-mediated CAF education downstream of ROCK. We find that CRELD2 is regulated by PERK-regulated ATF4, and depleting CRELD2 suppressed tumour progression, demonstrating that the paracrine ROCK-PERK-ATF4-CRELD2 axis promotes the progression of breast cancer, with implications for cancer therapy.

Nasopharyngeal carcinoma (NPC) mainly appears in southeastern Asian countries, including China. Adriamycin (ADM), a type of antitumor drug, is widely applied in treatments against various cancers. Nevertheless, cancer cells will eventually develop drug resistance to ADM. The present study aims to explore the potential role of reticulocalbin-1 (RCN1) in NPC cells resistance to ADM. Microarray-based analysis was used to screen NPC-related genes, with RCN1 acquired for this current study. RCN1 expression in NPC tissues and cells was determined. The biological function of RCN1 on NPC cell apoptosis was evaluated via gain- and loss-of-function experiments in 5–8 F/ADM and 5–8 F cells by delivering si-RCN1 and RCN1-vector. The function of endoplasmic reticulum (ER) stress on cell apoptosis was measured with the involvement of the PERK-CHOP signaling pathway. Furthermore, tumor formation in nude mice was performed to evaluate the survival condition and RCN1 effects in vivo. RCN1 was highly expressed in NPC tissues and cell lines. The increased expression of ER-related proteins ATF4, CHOP, and the extents of IRE1 and PERK phosphorylation were observed. RCN1 knockdown was found to reduce resistance of NPC cells/tissues to ADM while activating ER stress through the activated PERK-CHOP signaling pathway, which further promoted NPC cell apoptosis. These in vitro findings were detected in vivo on tumor formation in nude mice. In conclusion, the present study provides evidence that RCN1 knockdown stimulates ADM sensitivity in NPC by promoting ER stress-induced cell apoptosis, highlighting a theoretical basis for NPC treatment.

An overwhelming endoplasmic reticulum stress (ERS) and the following unfolded protein response (UPR) can induce hepatic inflammation, fibrosis and hepatocellular carcinoma (HCC). Caudatin, one of the species of C‑21 steroidal glycosides mainly isolated from the roots of Cynanchum bungei Decne, exhibits potent anticancer activities in vivo. However, the effect of caudatin on HCC remains unclear. In the present study, a diethylnitrosamine (DEN)‑induced HCC model was established. Nodules and tumors in rat livers were monitored by T2‑/T1‑weighted‑magnetic resonance imaging (MRI) using a 1.5 T scanner. Caudatin reduced the number and size of nodules and alleviated the inflammatory foci in the liver. In addition, the hepatic pro‑inflammatory levels of interleukin (IL) 6, monocyte chemoattractant protein 1 and IL‑1β were decreased in caudatin‑treated rats. The DEN‑induced surge in malondialdehyde, aspartate aminotransferase, alanine transaminase and TBIL were alleviated following caudatin treatment. The expression of ERS chaperones glucose‑regulated protein, 94 kDa, glucose‑regulated protein, 78 kDa and protein disulfide‑isomerase A4 and the proliferation marker Ki‑67 in liver nodules were all downregulated by caudatin as demonstrated by immunohistochemistry, reverse transcription‑quantitative PCR and western blot analysis. Caudatin reduced the cytoprotective ERS sensor activating transcription factor 6‑mediated signal transduction and inhibited the PKR‑like endoplasmic reticulum kinase/eukaryotic initiation factor 2α/activating transcription factor 4 pathway. However, the effect of caudatin on inositol requiring enzyme 1 signaling was negligible. In conclusion, restoration of the dysregulated UPR program was involved in the antitumor efficacy of caudatin without inducing cumulative hepatotoxicity.

Vitiligo is a depigmentation disorder that develops as a result of the progressive disappearance of epidermal melanocytes. The elevated level of amino acid metabolite homocysteine (Hcy) has been identified as circulating marker of oxidative stress and known as a risk factor for vitiligo. However, the mechanism underlying Hcy-regulated melanocytic destruction is currently unknown. The present study aims to elucidate the effect of Hcy on melanocytic destruction and its involvement in the pathogenesis of vitiligo. Our results showed that Hcy level was significantly elevated in the serum of progressive vitiligo patients. Notably, Hcy induced cell apoptosis in melanocytes via activating reactive oxygen species (ROS) and endoplasmic reticulum (ER) stress protein kinase RNA-like ER kinase (PERK)–eukaryotic translation initiation factor 2α (eIF2α)–C/EBP homologous protein (CHOP) pathway. More importantly, folic acid, functioning in the transformation of Hcy, could lower the intracellular Hcy level and further reverse the apoptotic effect of Hcy on melanocytes. Additionally, Hcy disrupted melanogenesis whereas folic acid supplementation could reverse the melanogenesis defect induced by Hcy in melanocytes. Taken together, Hcy is highly increased in vitiligo patients at progressive stage, and our in vitro studies revealed that folic acid could protect melanocytes from Hcy-induced apoptosis and melanin synthesis inhibition, indicating folic acid as a potential benefit agent for patients with progressive vitiligo.

Background and Purpose— Ischemic stroke impairs endoplasmic reticulum (ER) function, causes ER stress, and activates the unfolded protein response. The unfolded protein response consists of 3 branches controlled by ER stress sensor proteins, which include PERK (protein kinase RNA-like ER kinase). Activated PERK phosphorylates eIF2α (eukaryotic initiation factor 2 alpha), resulting in inhibition of global protein synthesis. Here, we aimed to clarify the role of the PERK unfolded protein response branch in stroke. Methods— Neuron-specific and tamoxifen-inducible PERK conditional knockout (cKO) mice were generated by cross-breeding Camk2a-CreERT2 with Perk f/f mice. Transient middle cerebral artery occlusion was used to induce stroke. Short- and long-term stroke outcomes were evaluated. Protein synthesis in the brain was assessed using a surface-sensing-of-translation approach. Results— After tamoxifen-induced deletion of Perk in forebrain neurons was confirmed in PERK-cKO mice, PERK-cKO and control mice were subjected to transient middle cerebral artery occlusion and 3 days or 3 weeks recovery. PERK-cKO mice had larger infarcts and worse neurological outcomes compared with control mice, suggesting that PERK-induced eIF2α phosphorylation and subsequent suppression of translation protects neurons from ischemic stress. Indeed, better stroke outcomes were observed in PERK-cKO mice that received postischemic treatment with salubrinal, which can restore the ischemia-induced increase in phosphorylated eIF2α in these mice. Finally, our data showed that post-treatment with salubrinal improved functional recovery after stroke. Conclusions— Here, we presented the first evidence that postischemic suppression of translation induced by PERK activation promotes recovery of neurological function after stroke. This confirms and further extends our previous observations that recovery of ER function impaired by ischemic stress critically contributes to stroke outcome. Therefore, future research should include strategies to improve stroke outcome by targeting unfolded protein response branches to restore protein homeostasis in neurons.

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.

Background: Increased keratinocyte proliferation occurs in the skin of psoriatic patients and is supposed to play a role in the pathogenesis of this disorder. Compounds interfering with keratinocyte proliferation could be useful in the management of psoriatic patients. Aim: To investigate whether albendazole, an anti-helmintic drug that regulates epithelial cell function in various systems, inhibits keratinocyte proliferation in models of psoriasis. Methods: Aldara-treated mice received daily topical application of albendazole. Keratinocyte proliferation and keratin (K) 6 and K16 expression were evaluated by immunohistochemistry and Western blotting and inflammatory cells/mediators were analysed by immunohistochemistry and real-time PCR. In human keratinocytes (HEKa and HaCaT) treated with albendazole, cell cycle and proliferation, keratins and cell cycle-associated factors were evaluated by flow cytometry, colorimetric assay and Western blotting respectively. Results: Aldara-treated mice given albendazole exhibited reduced epidermal thickness, decreased number of proliferating keratinocytes and K6/K16 expression. Reduction of CD3- and Ly6G-positive cells in the skin of albendazole-treated mice associated with inhibition of IL-6, TNF-α, IL-1β, IL-17A, IL-36, CCL17, CXCL1, CXCL2 and CXCL5 expression. Treatment of keratinocytes with albendazole reduced K6/K16 expression and reversibly inhibited cell growth by promoting accumulation of cells in S-phase. This phenomenon was accompanied by down-regulation of CDC25A, a phosphatase regulating progression of cell cycle through S-phase, and PKR-dependent hyper-phosphorylation of eIF2α, an inhibitor of CDC25 translation. In Aldara-treated mice, albendazole activated PKR, enhanced eIF2α phosphorylation and reduced CDC25A expression. Conclusions: Data show that albendazole inhibits keratinocyte proliferation and exerts therapeutic effect in a murine model of psoriasis.

As a branch of the unfolded protein response, protein kinase R-like endoplasmic reticulum kinase (PERK) represses global translation in response to endoplasmic reticulum (ER) stress. This pathophysiological condition is associated with the tumor microenvironment in cancer. Previous findings in our lab have suggested that PERK selectively represses translation of some mRNAs, but this possibility awaits additional investigation. In this study, we show that a stem-cell marker protein, leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5), is rapidly depleted in colon cancer cells during ER stress, an effect that depended on the PERK-mediated translational repression. Indeed, the PERK inhibition led to the accumulation of premature, underglycosylated forms of LGR5, which were produced only at low levels during proper PERK activation. Unlike the mature LGR5 form, which is constitutively degraded regardless of PERK activation, the underglycosylated LGR5 exhibited a prolonged half-life and accumulated inside the cells without being expressed on the cell surface. We also found that Erb-B2 receptor tyrosine kinase 3 (ERBB3) is subjected to a similarly-regulated depletion by PERK, whereas the epidermal growth factor receptor (EGFR), stress-inducible heat-shock protein family A (Hsp70) member 5 (HSPA5), and anterior gradient 2 protein-disulfide isomerase family member (AGR2) were relatively. insensitive to the PERK-mediated repression of translation. These results indicate that LGR5 and ERBB3 are targets for PERK-mediated translational repression during ER stress.

EIF2AK1 and EIF2AK2 encode members of the eukaryotic translation initiation factor 2 alpha kinase (EIF2AK) family that inhibits protein synthesis in response to physiologic stress conditions. EIF2AK2 is also involved in innate immune response and the regulation of signal transduction, apoptosis, cell proliferation, and differentiation. Despite these findings, human disorders associated with deleterious variants in EIF2AK1 and EIF2AK2 have not been reported. Here, we describe the identification of nine unrelated individuals with heterozygous de novo missense variants in EIF2AK1 (1/9) or EIF2AK2 (8/9). Features seen in these nine individuals include white matter alterations (9/9), developmental delay (9/9), impaired language (9/9), cognitive impairment (8/9), ataxia (6/9), dysarthria in probands with verbal ability (6/9), hypotonia (7/9), hypertonia (6/9), and involuntary movements (3/9). Individuals with EIF2AK2 variants also exhibit neurological regression in the setting of febrile illness or infection. We use mammalian cell lines and proband-derived fibroblasts to further confirm the pathogenicity of variants in these genes and found reduced kinase activity. EIF2AKs phosphorylate eukaryotic translation initiation factor 2 subunit 1 (EIF2S1, also known as EIF2α), which then inhibits EIF2B activity. Deleterious variants in genes encoding EIF2B proteins cause childhood ataxia with central nervous system hypomyelination/vanishing white matter (CACH/VWM), a leukodystrophy characterized by neurologic regression in the setting of febrile illness and other stressors. Our findings indicate that EIF2AK2 missense variants cause a neurodevelopmental syndrome that may share phenotypic and pathogenic mechanisms with CACH/VWM.

Protein kinase R (PKR) is a key antiviral component of the innate immune pathway and is activated by viral double-stranded RNAs (dsRNAs). Adenovirus-associated RNA 1 (VAI) is an abundant, noncoding viral RNA that functions as a decoy by binding PKR but not inducing activation, thereby inhibiting the antiviral response. In VAI, coaxial stacking produces an extended helix that mediates high-affinity PKR binding but is too short to result in activation. Like adenovirus, Epstein-Barr virus produces high concentrations of a noncoding RNA, EBER1. Here, we compare interactions of PKR with VAI and EBER1 and present a structural model of EBER1. Both RNAs function as inhibitors of dsRNA-mediated PKR activation. However, EBER1 weakly activates PKR whereas VAI does not. PKR binds EBER1 more weakly than VAI. Assays at physiological ion concentrations indicate that both RNAs can accommodate two PKR monomers and induce PKR dimerization. A structural model of EBER1 was obtained using constraints derived from chemical structure probing and small-angle X-ray scattering experiments. The central stem of EBER1 coaxially stacks with stem loop 4 and stem loop 1 to form an extended RNA duplex of ∼32 bp that binds PKR and promotes activation. Our observations that EBER1 binds PKR much more weakly than VAI and exhibits weak PKR activation suggest that EBER1 is less well suited to function as an RNA decoy.

Gene expression in mammalian cells results from coordinated protein-driven processes guided by diverse mechanisms of regulation, including protein-protein interactions, protein localization, DNA modifications and chromatin rearrangement. Regulation of gene expression is particularly important in stress-response pathways. To address the need to monitor chromosomal gene expression generating a readily detectable signal output that recapitulates gene expression dynamics, we developed a gene signal amplifier platform that links transcriptional and post-translational regulation of a fluorescent output to the expression of a chromosomal target gene. We generated a multiplex reporter system for monitoring markers of the unfolded protein response, a complex signal transduction pathway that remodels gene expression in response to proteotoxic stress in the endoplasmic reticulum. By recapitulating the transcriptional and translational control mechanisms underlying the expression of a target gene with high sensitivity, this platform provides a technology for monitoring gene expression with superior sensitivity and dynamic resolution.

OBJECTIVE Endoplasmic reticulum stress (ERS) is present in chondrocytes of osteoarthritis, and the intensity of ERS is related to the degree of cartilage degeneration. In vitro intervention strategies can change the status of ERS and induce the inhibition of ERS-related pathway. Therefore, this study is designed to explore the role and molecular mechanism of cartilage stem cells (ACSCs) of ERS in chondrocytes after hip replacement. METHODS Human cartilage cell lines C28/I2 were cultured as the control group. The ERS inducer was added into C28/I2 as ERS group. The third ERS + stem cells group was formed by adding cartilage stem cells into ERS group, and further transfection of si-PERK was defined as si-PERK + ERS + stem cells group. Cell cycle and apoptosis in the four groups were determined by flow cytometry. The protein expression of GRP78, PERK, ATF4, TMEM119, CDK4, Cyclin D, and BMP6 in chondrocytes in the four groups were investigated by western blot, and the distribution of PERK, TMEM119, and BMP6 in chondrocytes were observed by immunofluorescence assay. In addition, the transcriptional levels of Bcl2, Bax, and Caspase 3 were also determined by RT-PCR. RESULTS In cell cycle assay, ERS increased the accumulation of cells in G0 /G1 and G2 /M, while cartilage stem cells weakened the effects. The apoptosis rates in control group, ERS, ERS + stem cells, si-PERK + ERS + stem cells were 0%, 21.3%, 18.9%, and 15.9%, respectively, and the difference of apoptosis rate between the latter three groups and control group was statistically significant (P

Haploid genetic screening of cells under different types of mitochondrial perturbation shows that a pathway involving OMA1, DELE1 and the eIF2 alpha kinase HRI communicates mitochondrial stress to the cytosol to trigger the integrated stress response.Mitochondrial fidelity is tightly linked to overall cellular homeostasis and is compromised in ageing and various pathologies(1-3). Mitochondrial malfunction needs to be relayed to the cytosol, where an integrated stress response is triggered by the phosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2 alpha) in mammalian cells(4,5). eIF2 alpha phosphorylation is mediated by the four eIF2 alpha kinases GCN2, HRI, PERK and PKR, which are activated by diverse types of cellular stress(6). However, the machinery that communicates mitochondrial perturbation to the cytosol to trigger the integrated stress response remains unknown(1,2,7). Here we combine genome engineering and haploid genetics to unbiasedly identify genes that affect the induction of C/EBP homologous protein (CHOP), a key factor in the integrated stress response. We show that the mitochondrial protease OMA1 and the poorly characterized protein DELE1, together with HRI, constitute the missing pathway that is triggered by mitochondrial stress. Mechanistically, stress-induced activation of OMA1 causes DELE1 to be cleaved into a short form that accumulates in the cytosol, where it binds to and activates HRI via its C-terminal portion. Obstruction of this pathway can be beneficial or adverse depending on the type of mitochondrial perturbation. In addition to the core pathway components, our comparative genetic screening strategy identifies a suite of additional regulators. Together, these findings could be used to inform future strategies to modulate the cellular response to mitochondrial dysfunction in the context of human disease.

Epithelial ovarian cancer (EOC) is a leading cause of cancer-related mortality in the United States due to the late-stage disease at diagnosis. Overexpression of GRP78 and PDI following endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR) promote growth and invasion in cancer. To identify novel prognostic biomarkers in EOC, here we determined the expression of ER stress-associated proteins (GRP78, ATF6 and PERK) and correlated with clinical outcome in EOC. Tissue microarray (TMA) samples from 415 tissues collected from three cancer centers (UM, USC, and KCCRI) were used to assess the expression levels of ER-associated proteins using immunohistochemistry (IHC). We observed that the expression levels of GRP78 (p p p p p = 0.03), while low expression of combined GRP78 and PDI correlated with better survival (p = 0.01) in high-grade serous. The increased expression of ER stress-associated proteins in EOC suggests a role for ER stress and the UPR in EOC. More importantly, our results demonstrate that GRP78 and PDI are potential biomarkers for EOC and could be used as dual prognostic markers.

Sestrin2 (SESN2) is a highly evolutionary conserved protein and involved in different cellular responses to various stresses. However, the potential function of SESN2 in immune system remains unclear. The present study was designed to test whether dendritic cells (DCs) could express SESN2, and investigate the underlying molecular mechanism as well as its potential significance. Herein, we firstly reported that SESN2 was expressed in DCs after high mobility group box-1 protein (HMGB1) stimulation and the apoptosis of DCs was obviously increased when SESN2 gene silenced by siRNA. Cells undergone SESN2-knockdown promoted endoplasmic reticulum (ER) stress (ERS)-related cell death, markedly exacerbated ER disruption as well as the formation of dilated and aggregated structures, and they significantly aggravated the extent of ERS response. Conversely, overexpressing SESN2 DCs markedly decreased apoptotic rates and attenuated HMGB1-induced ER morphology fragment together with inhibition of ERS-related protein translation. Furthermore, sesn2−/−-deficient mice manifested increased DC apoptosis and aggravated ERS extent in septic model. These results indicate that SESN2 appears to be a potential regulator to inhibit apoptotic ERS signaling that exerts a protective effect on apoptosis of DCs in the setting of septic challenge.

Cancer cells exploit many of the cellular adaptive responses to support their survival needs. One of these is the unfolded protein response (UPR), a highly conserved signaling pathway that is mounted in response to endoplasmic reticulum (ER) stress. Recent work showed that steroid hormones, in particular estrogens and androgens, regulate the canonical UPR pathways in breast cancer (BCa) and prostate cancer (PCa). In addition, UPR has pleiotropic effects in advanced disease and development of therapy resistance. These findings implicate the UPR pathway as a novel target in hormonally regulated cancers in the clinic. Here, we review the potential therapeutic value of recently developed small molecule inhibitors of UPR in hormone regulated cancers.

Background: Acupuncture has been clinically recommended as a method of pain relief by the World Health Organization and is widely used by medical doctors. Fibromyalgia (FM) pain has a complex physiological and psychological origin and can be pharmacologically treated with duloxetine, milnacipran and pregabalin. However, these drugs produce undesirable side effects, such as headaches, nausea and diarrhoea. Acupuncture may serve as an effective alternative treatment for pain relief with few side effects. Aims: We hypothesised that acupuncture would reduce FM pain by influencing transient receptor potential cation channel subfamily V member 1 (TRPV1) and the downstream phosphorylated extracellular signal-regulated kinases (pERK), which are located in the central thalamus, amygdala and cortex. Methods: A FM mouse model was established by injecting two doses of acid saline into 32 female C57/B6 mice. The mice were then assigned to different subgroups (n=8 each) and treated with electroacupuncture (EA) or EA sham control. TRPV1 and pERK expression levels were measured using Western blotting and immunohistochemistry. Results: Our results demonstrated that the expression of TRPV1 and pERK in the thalamus, amygdala and somatosensory cortex was normal in the control mice, but significantly increased in FM mice; these FM-induced changes in expression were attenuated by EA. Conclusion: Our data suggest that EA can reverse the central sensitisation of the TRPV1-ERK signalling pathway in the mouse brain. Thus, our findings provide mechanistic evidence supporting the potential therapeutic efficacy of EA for treating FM pain.

Background: Diabetes diagnosed within the first 6 months of life is defined as neonatal diabetes mellitus (NDM). Mutations in the KCNJ11, ABCC8, and INS genes are the most common cause of permanent NDM. In populations with a high rate of consanguinity, Wolcott-Rallison syndrome caused by biallelic EIF2AK3 mutations is common. Methods: We studied the clinical characteristics and underlying genetic cause of disease in 15 individuals with diabetes onset before 6 months of age as defined by sustained hyperglycaemia requiring insulin treatment. Patients who had a remission of the diabetes, defined by a normal blood glucose and HbA1c value without insulin or sulphonylurea (SU) treatment, within the first 18 months of life were classified as having transient NDM (TNDM). Results: We report 15 patients with NDM from 14 unrelated families, including 10 with reported parental consanguinity. 1/15 patients had a remission of diabetes, leading to a diagnosis of TNDM. Mutations were detected in 80% (n = 12/15) of the cohort (ABCC8 [n = 4], PTF1A-distal enhancer [n = 3], KCNJ11 [n = 2], EIF2AK3 [n = 1], INS [n = 1], and SLC19A2 [n = 1]). All cases were initially treated with multiple dose insulin injections. One patient with an ABCC8 mutation transitioned from insulin to SU resulting in improved metabolic control at the age of 20 years. Conclusion: Although the number of individuals born to consanguineous parents was considerably high in this cohort, KATP channel mutations (ABCC8/KCNJ11) were more common than EIF2AK3 mutations (n = 6 vs. n = 1). Genetic analyses should be performed in all NDM cases due to the potential impact on treatment and prognosis.

Venezuelan equine encephalitis virus (VEEV) is a neurotropic virus that causes significant disease in both humans and equines. Here we characterized the impact of VEEV on signaling pathways regulating cell death in human primary astrocytes. VEEV productively infected primary astrocytes and caused an upregulation of early growth response 1 (EGR1) gene expression at 9 and 18 h post infection. EGR1 induction was dependent on extracellular signal-regulated kinase1/2 (ERK1/2) and protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), but not on p38 mitogen activated protein kinase (MAPK) or phosphoinositide 3-kinase (PI3K) signaling. Knockdown of EGR1 significantly reduced VEEV-induced apoptosis and impacted viral replication. Knockdown of ERK1/2 or PERK significantly reduced EGR1 gene expression, dramatically reduced viral replication, and increased cell survival as well as rescued cells from VEEV-induced apoptosis. These data indicate that EGR1 activation and subsequent cell death are regulated through ERK and PERK pathways in VEEV infected primary astrocytes.

Growth hormone (GH) has multiple physiological roles, acting on many organs. In order to investigate its roles in rat liver, we tried to identify novel genes whose transcription was regulated by GH. We identified X-box binding protein 1 (Xbp1) as a candidate gene. XBP1 is a key transcription factor activated in response to endoplasmic reticulum (ER) stress. The purpose of this study was to investigate the mode of action of GH on XBP1, including the relation with ER stress, sex-dependent expression of the mRNA, and the signaling pathway. Intravenous administration of GH rapidly and transiently increased Xbp1 mRNA in hypophysectomized rat livers. Neither phosphorylated inositol-requiring-1α (IRE1α) nor phosphorylated PKR-like ER kinase (PERK) increased, suggesting that Xbp1 expression is induced by an ER stress-independent mechanism. The active form of XBP1(S) protein was increased by GH administration and was followed by an increased ER-associated dnaJ protein 4 (ERdj4) mRNA level. XBP1(S) protein levels were predominantly identified in male rat livers with variations among individuals similar to those of phosphorylated signal transducer and activator of transcription 5B (STAT5B), suggesting that XBP1(S) protein levels are regulated by the sex-dependent secretary pattern of GH. The GH signaling pathway to induce Xbp1 mRNA was examined in rat hepatoma H4IIE cells. GH induced the phosphorylation of CCAAT/enhancer-binding protein β (C/EBPβ) following extracellular signal-regulated protein kinase (ERK) phosphorylation. Taken together, the results indicated that XBP1 is activated by GH in rat liver in a sexually dimorphic manner via ERK and C/EBPβ pathway.

Asparaginase (ASNase) causes pancreatitis in approximately 10% of leukemia patients, and the mechanisms underlying this painful complication are not known. ASNase primarily depletes circulating asparagine, and the endogenously expressed enzyme, asparagine synthetase (ASNS), replenishes asparagine. ASNS was suggested previously to be highly expressed in the pancreas. In this study, we determined the expression pattern of ASNS in the pancreas and the mechanism for increased pancreatic ASNS abundance. Compared with other organs, ASNS was highly expressed in both the human and mouse pancreas, and, within the pancreas, ASNS was present primarily in the acinar cells. The high baseline pancreatic ASNS was associated with higher baseline activation of protein kinase R-like endoplasmic reticulum kinase (PERK) signaling in the pancreas, and inhibition of PERK in acinar cells lessened ASNS expression. ASNase exposure, but not the common pancreatitis triggers, uniquely up-regulated ASNS expression, indicating that the increase is mediated by nutrient stress. The up-regulation of acinar ASNS with ASNase exposure was owing to increased transcriptional rather than delayed degradation. Knockdown of ASNS in the 266-6 acinar cells provoked acinar cell injury and worsened ASNase-induced injury, whereas ASNS overexpression protected against ASNase-induced injury. In summary, ASNS is highly expressed in the pancreatic acinar cells through heightened basal activation of PERK, and ASNS appears to be crucial to maintaining acinar cell integrity. The implications are that ASNS is especially hardwired in the pancreas to protect against both baseline perturbations and nutrient deprivation stressors, such as during ASNase exposure. Keywords: Leukemia, Asparaginase-Associated Pancreatitis, Asparagine Synthetase, PERK Signaling

Background Circular RNAs (circRNAs) represent a novel type of regulatory RNA characterized by high evolutionary conservation and stability. CircRNAs are expected to be potential diagnostic biomarkers and therapeutic targets for a variety of malignancies. However, the regulatory functions and underlying mechanisms of circRNAs in triple-negative breast cancer (TNBC) are largely unknown. Methods By using RNA high-throughput sequencing technology, qRT-PCR and in situ hybridization assays, we screened dysregulated circRNAs in breast cancer and TNBC tissues. Then in vitro assays, animal models and patient-derived organoids (PDOs) were utilized to explore the roles of the candidate circRNA in TNBC. To investigate the underlying mechanisms, RNA pull-down, RNA immunoprecipitation (RIP), co immunoprecipitation (co-IP) and Western blotting assays were carried out. Results In this study, we demonstrated that circRNA-CREIT was aberrantly downregulated in doxorubicin resistant triple-negative breast cancer (TNBC) cells and associated with a poor prognosis. The RNA binding protein DHX9 was responsible for the reduction in circRNA-CREIT by interacting with the flanking inverted repeat Alu (IRAlu) sequences and inhibiting back-splicing. By utilizing in vitro assays, animal models and patient-derived organoids, we revealed that circRNA-CREIT overexpression significantly enhanced the doxorubicin sensitivity of TNBC cells. Mechanistically, circRNA-CREIT acted as a scaffold to facilitate the interaction between PKR and the E3 ligase HACE1 and promoted proteasomal degradation of PKR protein via K48-linked polyubiquitylation. A reduced PKR/eIF2α signaling axis was identified as a critical downstream effector of circRNA-CREIT, which attenuated the assembly of stress granules (SGs) to activate the RACK1/MTK1 apoptosis signaling pathway. Further investigations revealed that a combination of the SG inhibitor ISRIB and doxorubicin synergistically inhibited TNBC tumor growth. Besides, circRNA-CREIT could be packaged into exosomes and disseminate doxorubicin sensitivity among TNBC cells. Conclusions Our study demonstrated that targeting circRNA-CREIT and SGs could serve as promising therapeutic strategies against TNBC chemoresistance.

Vanadium (V) is a common environmental and industrial pollutant that can cause nephrotoxicity in animals in excess. The purpose of this research was to explore the interaction between endoplasmic reticulum (ER) stress and autophagy induced by V in the kidney of ducks. Duck renal tubule epithelial cells were exposed to different concentrations of sodium metavanadate (NaVO

Delirium is the most common postoperative complication in older patients after prolonged anesthesia and surgery and is associated with accelerated cognitive decline and dementia. The neuronal pathogenesis of postoperative delirium is largely unknown. The unfolded protein response (UPR) is an adaptive reaction of cells to perturbations in endoplasmic reticulum function. Dysregulation of UPR has been implicated in a variety of diseases including Alzheimer's disease and related dementias. However, whether UPR plays a role in anesthesia-induced cognitive impairment remains unexplored. By performing in vivo calcium imaging in the mouse frontal cortex, we showed that exposure of aged mice to the inhalational anesthetic sevoflurane for 2 hours resulted in a marked elevation of neuronal activity during recovery, which lasted for at least 24 hours after the end of exposure. Concomitantly, sevoflurane anesthesia caused a prolonged increase in phosphorylation of PERK and eIF2α, the markers of UPR activation. Genetic deletion or pharmacological inhibition of PERK prevented neuronal hyperactivity and memory impairment induced by sevoflurane. Moreover, we showed that PERK suppression also reversed various molecular and synaptic changes induced by sevoflurane anesthesia, including alterations of synaptic NMDA receptors, tau protein phosphorylation, and dendritic spine loss. Together, these findings suggest that sevoflurane anesthesia causes abnormal UPR in the aged brain, which contributes to neuronal hyperactivity, synapse loss and cognitive decline in aged mice.

The spleen is an important site of hematopoietic stem/progenitor cell (HSPC) preconditioning and tumor-promoting myeloid cell generation in cancer, but the regulatory mechanism remains unclear. Here, we found that PKR-like endoplasmic reticulum kinase (PERK) mediated HSPC reprogramming into committed MDSC precursors in the spleen via PERK–ATF4–C/EBPβ signaling. Pharmacological and genetic inhibition of this pathway in murine and human HSPCs prevented their myeloid descendant cells from becoming MDSCs even with subsequent exposure to tumor microenvironment (TME) factors. In mice, the selective delivery of PERK antagonists to the spleen was not only sufficient but more effective than the tumor-targeted strategy in preventing MDSC activation in the tumor, leading to profound TME reshaping and tumor regression. Clinically, HSPCs in the spleen of cancer patients exhibit increased PERK signaling correlated with enhanced myelopoiesis. Our findings indicate that PERK-mediated HSPC preconditioning plays a crucial role in MDSC generation, suggesting novel spleen-targeting therapeutic opportunities for restraining the tumor-promoting myeloid response at its source.

Hyperglycemia induced reactive oxygen species oxidize macromolecules including cellular proteins leading to their accumulation in Endoplasmic Reticulum (ER) lumen which in turn activates unfolded protein response (UPR) sensors including, PERK (Protein Kinase RNA-Like ER Kinase). Activated PERK induces ER associated degradation of misfolded proteins to lower the ER stress. In the present study, we hypothesized that ER stress leads to the degradation of glucose transporter proteins resulting in complex glucose metabolism. In vivo studies were carried out in the experimental model of hyperglycemia using streptozotocin/nicotinamide induced diabetic male Wistar rats. High glucose (30 mM) treated HepG2 cells were used to perform the mechanistic study at different time points. PERK gene knockdown (siRNA transfection) and inhibition by ISRIB (Integrated Stress Response Inhibitor, a potent inhibitor of PERK signaling) confirmed the involvement of PERK axis in regulating the expression and translocation of hepatic glucose transporters. Co-immunoprecipitation and dual immunostaining studies further demonstrated increased degradation of GLUT proteins under high glucose conditions. Moreover, Morin (3,5,7,2',4' pentahydroxyflavone) treatment prevented PERK-eIF2α-ATF4 mediated degradation of glucose transporters and enhanced glucose uptake in both, HepG2 cells and diabetic rats. Targeting aberrant regulation of the expression and translocation of facilitative glucose transporter proteins (GLUT proteins) may provide novel therapeutic strategies for the better management of diabetes.

Several epidemiological studies have reported significant associations between prenatal polybrominated diphenyl ethers (PBDEs) exposure and adverse birth outcomes. Placental injury is thought to mediate these associations. However, few study has investigated the adverse effects of PBDEs exposure on placental growth and development. We examined the impacts of gestational exposure to BDE-209, the most abundant PBDE conger detected in human samples, on placental structure and function, and its model of action in vivo and in vitro. Pregnant mice were exposed to 0, 2, 20, 200 mg/kg/day of BDE-209 by gavages from gestational day (GD) 0 to GD18. Results showed that gestational BDE-209 exposure significantly reduced placental weight, impaired placental vascular development and induced placental cell apoptosis. In addition, gestational BDE-209 exposure impaired placental transport and endocrine function as demonstrated by markedly downregulated expression of Glut1, Znt1, Pgf and Igf2 in BDE-209-treated placentas. Mechanistically, gestational exposure to BDE-209 upregulated the expression of GRP78, and 3 downstream proteins (p-eIF2α, ATF4 and CHOP) of the PERK signaling, suggesting the activation of endoplasmic reticulum (ER) stress and PERK signaling pathway in mouse placentas. Further in vitro study showed that PERK siRNA pretreatment markedly reversed BDE-209-induced cell apoptosis in human JEG-3 cells. Collectively, our results suggest that the activation of the ER stress-mediated PERK/ATF4/CHOP signaling pathway played a role in BDE-209-induced placental injury. Our findings provide new insight into the mechanisms of BDE-209 induced reproductive and developmental toxicity.

Melatonin exhibits numerous anti-cancer activities in the treatment of human cancers. Nevertheless, the mechanisms of anti-gastric cancer effect of melatonin is still unclear. The aim of the study is to investigate the interaction between melatonin, endoplasmic reticulum (ER) stress, NF-κB signaling and HSF1 protein in gastric cancer cells.In the current study, we used CCK-8, flow cytometry and Western blot to research anticancer mechanism of melatonin in AGS cells.The data demonstrated that melatonin could suppress cell proliferation and increase cell apoptosis. We explore that the ER stress and NF-kB signaling pathways play crucial roles in the cell apoptosis process. Of note, melatonin increased the expression of p-PERK and p-eIF2α, and decreased the expression of p-P65 and p-IκBα. A combination of melatonin and PERK inhibitor (GSK2606414) or NF-κB inhibitor (Bay11-7082) suppressed the activation PERK/eIF2α and NF-κB signaling pathway. Subsequently, the expression of HSF1 protein was upregulated by melatonin and kept its expression by Bay 11-7082.These results suggest that melatonin induces AGS cell apoptosis by up-regulating PERK/eIF2α and downregulating NF-κB signaling pathway.

Retinal pigment epithelium (RPE) performs essential functions for ensuring retinal homeostasis and is a key site for pathogenic changes leading to age-related macular degeneration (AMD). Compromised proteostasis in RPE results in ER stress and ER stress-dependent antioxidant, apoptosis and autophagic responses. ER stress induces the unfolded protein response (UPR) in which EIF2AK3, encoding the protein kinase RNA-like ER kinase (PERK), acts as a key regulator. Downregulated EIF2AK3 gene expression has recently been identified in AMD using human donor RPE, however the molecular mechanisms that integrate the various ER-mediated cellular pathways underpinning progressive RPE dysfunction in AMD have not been fully characterised. This study investigated the downstream effects of PERK downregulation in response to Brefeldin A (BFA)-induced ER stress in ARPE-19 cells. PERK downregulation resulted in increased ER stress and impaired apoptosis induction, antioxidant responses and autophagic flux. ARPE-19 cells were unable to efficiently induce autophagy following PERK downregulation and PERK presented a role in regulating the rate of autophagy induction. The findings support PERK downregulation as an integrative event facilitating dysregulation of RPE processes critical to cell survival known to contribute to AMD development and highlight PERK as a potential future therapeutic target for AMD.

Control of protein synthesis (mRNA translation) plays key roles in shaping the proteome and in many physiological, including homeostatic, responses. One long-known translational control mechanism involves phosphorylation of initiation factor, eIF2, which is catalysed by any one of four protein kinases, which are generally activated in response to stresses. They form a key arm of the integrated stress response (ISR). Phosphorylated eIF2 inhibits eIF2B (the protein that promotes exchange of eIF2-bound GDP for GTP) and thus impairs general protein synthesis. However, this mechanism actually promotes translation of certain mRNAs by virtue of specific features they possess. Recent work has uncovered many previously unknown features of this regulatory system. Several studies have yielded crucial insights into the structure and control of eIF2, including that eIF2B is regulated by several metabolites. Recent studies also reveal that control of eIF2 and the ISR helps determine organismal lifespan and surprising roles in sensing mitochondrial stresses and in controlling the mammalian target of rapamycin (mTOR). The latter effect involves an unexpected role for one of the eIF2 kinases, HRI. Phosphoproteomic analysis identified new substrates for another eIF2 kinase, Gcn2, which senses the availability of amino acids. Several genetic disorders arise from mutations in genes for eIF2α kinases or eIF2B (i.e. vanishing white matter disease, VWM and microcephaly, epileptic seizures, microcephaly, hypogenitalism, diabetes and obesity, MEHMO). Furthermore, the eIF2-mediated ISR plays roles in cognitive decline associated with Alzheimer's disease. New findings suggest potential therapeutic value in interfering with the ISR in certain settings, including VWM, for example by using compounds that promote eIF2B activity.

Diarrhea-predominant irritable bowel syndrome (IBS-D) is a common functional gastrointestinal disease. Tong-Xie-Yao-Fang (TXYF), the traditional Chinese herbal medicine prescription, is a classic and effective prescription for the treatment of IBS-D, but its mechanism of action is not fully clarified.To evaluate the efficacy of TXYF in the treatment of IBS-D and to explore its potential mechanism of action.Changes in the serum levels of 50 free amino acids were targeted for detection by high-performance liquid chromatography (HPLC), and the expression of glucose-regulated protein 78 (GRP78), general control nonderepressible 2 (GCN2), and endoplasmic reticulum-resident kinase (PERK) was detected by immunohistochemistry examinations in healthy volunteers and IBS-D patients. The IBS-D rat was constructed by the three-factor superposition method of neonatal maternal separation, 2,4,6-trinitrobenzene sulfonic acid enema, and chronic unpredictable stress stimulation. The treatment effect of TXYF on IBS-D rats was observed by recording the body weight, grasp force, fecal water content (FWC), and abdominal withdrawal reflex (AWR) of rats before and after treatment. The effects of GCN2/PERK-eukaryotic initiation factor-2 (eIF2α) -activating transcription Factor 4 (ATF4) pathway proteins and gene expression were analyzed by western blotting, reverse transcription-polymerase chain reaction (RT-qPCR), and immunohistochemistry evaluations.Compared with healthy volunteers, IBS-D patients exhibited lower levels of cysteine, γ-aminoacetic acid (GABA), homoproline, and lysine, and immunohistochemistry showed strong activation of GRP78, a marker of endoplasmic reticulum stress. Differential expression of GCN2 and PERK proteins was detected in IBS-D patients and rat colons. In the IBS-D rats, TXYF improved the body weight and grasp force, reduced the FWC, and improved the AWR score. TXYF increased the levels of p-GCN2 and GCN2 and reduced the levels of GRP78, p-PERK, PERK, p-eIF2α, and eIF2α, thereby affecting the expression of the apoptosis-related transcription factors ATF4, CHOP, Caspase-3, and Bcl-2.Our study showed that TXYF improved IBS-D by inhibiting apoptosis. The anti-apoptosis effects were potentially mediated by regulating the GCN2/PERK-eIF2a-ATF4 signaling pathway.

Proteasome dysfunction can lead to autoinflammatory disease associated with elevated type I interferon (IFN-αβ) and NF-κB signaling; however, the innate immune pathway driving this is currently unknown. Here, we identified protein kinase R (PKR) as an innate immune sensor for proteotoxic stress. PKR activation was observed in cellular models of decreased proteasome function and in multiple cell types from patients with proteasome-associated autoinflammatory disease (PRAAS). Furthermore, genetic deletion or small-molecule inhibition of PKR in vitro ameliorated inflammation driven by proteasome deficiency. In vivo, proteasome inhibitor–induced inflammatory gene transcription was blunted in PKR-deficient mice compared with littermate controls. PKR also acted as a rheostat for proteotoxic stress by triggering phosphorylation of eIF2α, which can prevent the translation of new proteins to restore homeostasis. Although traditionally known as a sensor of RNA, under conditions of proteasome dysfunction, PKR sensed the cytoplasmic accumulation of a known interactor, interleukin-24 (IL-24). When misfolded IL-24 egress into the cytosol was blocked by inhibition of the endoplasmic reticulum–associated degradation pathway, PKR activation and subsequent inflammatory signaling were blunted. Cytokines such as IL-24 are normally secreted from cells; therefore, cytoplasmic accumulation of IL-24 represents an internal danger-associated molecular pattern. Thus, we have identified a mechanism by which proteotoxic stress is detected, causing inflammation observed in the disease PRAAS.

Activation of unfolded protein responses (UPRs) in cancer cells undergoing endoplasmic reticulum (ER) stress promotes survival. However, how UPR in tumor cells impacts anti-tumor immune responses remains poorly described. Here, we investigate the role of the UPR mediator pancreatic ER kinase (PKR)-like ER kinase (PERK) in cancer cells in the modulation of anti-tumor immunity. Deletion of PERK in cancer cells or pharmacological inhibition of PERK in melanoma-bearing mice incites robust activation of anti-tumor T cell immunity and attenuates tumor growth. PERK elimination in ER-stressed malignant cells triggers SEC61β-induced paraptosis, thereby promoting immunogenic cell death (ICD) and systemic anti-tumor responses. ICD induction in PERK-ablated tumors stimulates type I interferon production in dendritic cells (DCs), which primes CCR2-dependent tumor trafficking of common-monocytic precursors and their intra-tumor commitment into monocytic-lineage inflammatory Ly6C

Sec62 is a membrane protein of the endoplasmic reticulum that facilitates protein transport. Its role in cancer is increasingly recognised, but remains largely unknown. We investigated the functional role of Sec62 in gastric cancer (GC) and its underlying mechanism.Bioinformatics, tissue microarray, immunohistochemistry (IHC), western blotting (WB), quantitative polymerase chain reaction (qPCR), and immunofluorescence were used to examine the expression of target genes. Transwell, scratch healing assays, and xenograft models were used to evaluate cell migration and invasion. Transmission electron microscopy and mRFP-GFP-LC3 double-labeled adenoviruses were used to monitor autophagy. Co-immunoprecipitation (CO-IP) was performed to evaluate the binding activity between the proteins.Sec62 expression was upregulated in GC, and Sec62 upregulation was an independent predictor of poor prognosis. Sec62 overexpression promoted GC cell migration and invasion both in vitro and in vivo. Sec62 promoted migration and invasion by affecting TIMP-1 and MMP2/9 balance. Moreover, Sec62 could activate autophagy by upregulating PERK/ATF4 expression and binding to LC3II with concomitant FIP200/Beclin-1/Atg5 activation. Furthermore, autophagy blockage impaired the promotive effects of Sec62 on GC cell migration and invasion, whereas autophagy activation rescued the inhibitory effect of Sec62 knockdown on GC metastasis. Notably, Sec62 inhibition combined with autophagy blockage exerted a synergetic anti-metastatic effect in vitro and in vivo.Sec62 promotes GC metastasis by activating autophagy and subsequently regulating TIMP-1 and MMP2/9 balance. The activation of autophagy by Sec62 may involve the unfolded protein response (UPR)-related PERK/ATF4 pathway and binding of LC3II during UPR recovery involving FIP200/Beclin-1/Atg5 upregulation. Specifically, the dual inhibition of Sec62 and autophagy may provide a promising therapeutic strategy for GC metastasis.

This study aims to explore the crosstalk between GRP78/PERK/ATF-4 signaling pathway and renal apoptosis induced by nephropathogenic infectious bronchitis virus (NIBV). Hy-Line brown chickens were divided into two groups (Con, n = 100 and Dis, n = 200). At 28 days of age, each chicken in the Dis group was intranasally injected with SX9 strain (10(−5)/0.2 ml). Venous blood and kidney tissues were collected at 1, 5, 11, 18 and 28 days postinfection. Our results showed that NIBV infection upregulated the levels of creatinine, uric acid, and calcium (Ca(2+)) levels. Histopathological examination revealed severe hemorrhage and inflammatory cell infiltration near the renal tubules. Meanwhile, NIBV virus particles and apoptotic bodies were observed by ultramicro electron microscope. In addition, RT-qPCR and Western blot showed that NIBV upregulated the expression of GRP78, PERK, eIF2α, ATF-4, CHOP, Caspase-3, Caspase-9, P53, Bax, and on the contrary, downregulated the expression of Bcl-2. Furthermore, immunofluorescence localization analysis showed that the positive expression of Bcl-2 protein was significantly decreased. Correlation analysis indicated that endoplasmic reticulum (ER) stress gene expression, apoptosis gene expression, and renal injury were potentially related. Taken together, NIBV infection can induce renal ER stress and apoptosis by activating of GRP78/PERK/ATF-4 signaling pathway, leading to kidney damage. IMPORTANCE Nephropathogenic infectious bronchitis virus (NIBV) induced renal endoplasmic reticulum stress in chickens. NIBV infection induced kidney apoptosis in chickens. GRP78/PERK/ATF-4 signaling pathway is potentially related to renal apoptosis induced by NIBV.

HRI is the heme-regulated elF2α kinase that phosphorylates the α-subunit of elF2. Although the role of HRI in inhibiting globin synthesis in erythroid cells is well established, broader roles of HRI in translation have been uncovered recently. This review is to summarize the new discoveries of HRI in stress erythropoiesis and in fetal γ-globin expression.HRI and activating transcription factor 4 (ATF4) mRNAs are highly expressed in early erythroblasts. Inhibition of protein synthesis by HRI-phosphorylated elF2α (elF2αP) is necessary to maintain protein homeostasis in both the cytoplasm and mitochondria. In addition, HRI-elF2αP specifically enhances translation of ATF4 mRNA leading to the repression of mechanistic target of rapamycin complex 1 (mTORC1) signaling. ATF4-target genes are most highly activated during iron deficiency to maintain mitochondrial function, redox homeostasis, and to enable erythroid differentiation. HRI is therefore a master translation regulator of erythropoiesis sensing intracellular heme concentrations and oxidative stress for effective erythropoiesis. Intriguingly, HRI-elF2αP-ATF4 signaling also inhibits fetal hemoglobin production in human erythroid cells.The primary function of HRI is to maintain protein homeostasis accompanied by the induction of ATF4 to mitigate stress. Role of HRI-ATF4 in γ-globin expression raises the potential of HRI as a therapeutic target for hemoglobinopathy.

In response to endoplasmic reticulum (ER) stress, activation of pancreatic ER kinase (PERK) signaling adapts cells to stressful conditions by phosphorylating eukaryotic translation initiation factor 2α (eIF2α). Phosphorylation of eIF2α inhibits global protein translation but stimulates the expression of numerous stress-responsive genes by inducing the transcription factor ATF4. A large number of studies have shown that activation of PERK signaling has beneficial or detrimental effects in various diseases of the central nervous system (CNS), including neurodegenerative diseases, myelin disorders, CNS injuries, among others. This chapter is devoted to describing the practical methods for the detection of PERK signaling in CNS diseases.

Endoplasmic reticulum (ER) stress triggers an adaptive response in tumours which fosters cell survival and resilience to stress. Activation of the ER stress response, through its PERK branch, promotes phosphorylation of the α-subunit of the translation initiation factor eIF2, thereby repressing general protein translation and augmenting the translation of ATF4 with the downstream CHOP transcription factor and the protein disulfide oxidase, ERO1-alpha EXPERIMENTAL APPROACH: Here, we show that ISRIB, a small molecule that inhibits the action of phosphorylated eIF2alpha, activating protein translation, synergistically interacts with the genetic deficiency of protein disulfide oxidase ERO1-alpha, enfeebling breast tumour growth and spread.ISRIB represses the CHOP signal, but does not inhibit ERO1. Mechanistically, ISRIB increases the ER protein load with a marked perturbing effect on ERO1-deficient triple-negative breast cancer cells, which display impaired proteostasis and have adapted to a low client protein load in hypoxia, and ERO1 deficiency impairs VEGF-dependent angiogenesis. ERO1-deficient triple-negative breast cancer xenografts have an augmented ER stress response and its PERK branch. ISRIB acts synergistically with ERO1 deficiency, inhibiting the growth of triple-negative breast cancer xenografts by impairing proliferation and angiogenesis.These results demonstrate that ISRIB together with ERO1 deficiency synergistically shatter the PERK-dependent adaptive ER stress response, by restarting protein synthesis in the setting of impaired proteostasis, finally promoting tumour cytotoxicity. Our findings suggest two surprising features in breast tumours: ERO1 is not regulated via CHOP under hypoxic conditions, and ISRIB offers a therapeutic option to efficiently inhibit tumour progression in conditions of impaired proteostasis.

Neurological disorders are often progressive and lead to disabilities with limited available therapies. Epidemiological evidence implicated that prolonged exposure to hypoxia leads to neurological damage and a plethora of complications. Neural stem cells (NSCs) are a promising tool for neurological damage therapy in terms of their unique properties. However, the literature on the outcome of NSCs exposed to severe hypoxia is scarce. In this study, we identified a responsive gene that reacts to multiple cellular stresses, marked cold-inducible RNA-binding protein (CIRBP), which could attenuate NSC apoptosis under hypoxic pressure. Interestingly, ISRIB, a small-molecule modulator of the PERK-ATF4 signaling pathway, could prevent the reduction and apoptosis of NSCs in two steps: enhancing the expression of CIRBP through the protein kinase R- (PKR-) like endoplasmic reticulum kinase (PERK) and activating transcription factor 4 (ATF4) axis. Taken together, CIRBP was found to be a critical factor that could protect NSCs against apoptosis induced by hypoxia, and ISRIB could be acted upstream of the axis and may be recruited as an open potential therapeutic strategy to prevent or treat hypoxia-induced brain hazards.

Mesangial cells are modified smooth muscle cells with the ability to modulate glomerular filtration rate (GFR) - a marker of ischemic renal injury. We aimed to determine the role of intracellular O-GlcNAc levels and ER stress in mesangial cells subjected to ATP depletion. Immortalized mouse mesangial cells culture was incubated for 30, 45 and 60 min, or not (control group) with a buffer containing antimycin A and 2-deoxy-d-glucose, inhibitors of ATP synthesis. Mesangial cells subjected to ATPdepletion for 45 min followed by 24 h reperfusion (H

Deregulation of protein synthesis and ER stress/unfolded protein response (ER stress/UPR) have been reported in astrocytes. However, the relationships between protein synthesis deregulation and ER stress/UPR, as well as their role in the altered homeostatic support of Alzheimer’s disease (AD) astrocytes remain poorly understood. Previously, we reported that in astrocytic cell lines from 3xTg-AD mice (3Tg-iAstro) protein synthesis was impaired and ER-mitochondria distance was reduced. Here we show that impaired protein synthesis in 3Tg-iAstro is associated with an increase of p-eIF2α and downregulation of GADD34. Although mRNA levels of ER stress/UPR markers were increased two-three-fold, we found neither activation of PERK nor downstream induction of ATF4 protein. Strikingly, the overexpression of a synthetic ER-mitochondrial linker (EML) resulted in a reduced protein synthesis and augmented p-eIF2α without any effect on ER stress/UPR marker genes. In vivo, in hippocampi of 3xTg-AD mice, reduced protein synthesis, increased p-eIF2α and downregulated GADD34 protein were found, while no increase of p-PERK or ATF4 proteins was observed, suggesting that in AD astrocytes, both in vitro and in vivo, phosphorylation of eIF2α and impairment of protein synthesis are PERK-independent. Next, we investigated the ability of 3xTg-AD astrocytes to support metabolism and function of other cells of the central nervous system. Astrocyte conditioned medium (ACM) from 3Tg-iAstro cells significantly reduced protein synthesis rate in primary hippocampal neurons. When added as a part of pericyte/endothelial cell (EC)/astrocyte 3D co-culture, 3Tg-iAstro, but not WT-iAstro, severely impaired formation and ramification of tubules, the effect, replicated by EML overexpression in WT-iAstro cells. Finally, a chemical chaperone 4-phenylbutyric acid (4-PBA) rescued protein synthesis, p-eIF2α levels in 3Tg-iAstro cells and tubulogenesis in pericyte/EC/3Tg-iAstro co-culture. Collectively, our results suggest that a PERK-independent, p-eIF2α-associated impairment of protein synthesis compromises astrocytic homeostatic functions, and this may be caused by the altered ER-mitochondria interaction.

Patients suffering from immune-mediated necrotizing myopathies (IMNM) harbor, the pathognomonic myositis-specific auto-antibodies anti-SRP54 or -HMGCR, while about one third of them do not. Activation of chaperone-assisted autophagy was described as being part of the molecular etiology of IMNM. Endoplasmic reticulum (ER)/sarcoplasmic reticulum (SR)-stress accompanied by activation of the unfolded protein response (UPR) often precedes activation of the protein clearance machinery and represents a cellular defense mechanism toward restoration of proteostasis. Here, we show that ER/SR-stress may be part of the molecular etiology of IMNM. To address this assumption, ER/SR-stress related key players covering the three known branches (PERK-mediated, IRE1-mediated, and ATF6-mediated) were investigated on both, the transcript and the protein levels utilizing 39 muscle biopsy specimens derived from IMNM-patients. Our results demonstrate an activation of all three UPR-branches in IMNM, which most likely precedes the activation of the protein clearance machinery. In detail, we identified increased phosphorylation of PERK and eIF2a along with increased expression and protein abundance of ATF4, all well-documented characteristics for the activation of the UPR. Further, we identified increased general XBP1-level, and elevated XBP1 protein levels. Additionally, our transcript studies revealed an increased ATF6-expression, which was confirmed by immunostaining studies indicating a myonuclear translocation of the cleaved ATF6-form toward the forced transcription of UPR-related chaperones. In accordance with that, our data demonstrate an increase of downstream factors including ER/SR co-chaperones and chaperones (e.g., SIL1) indicating an UPR-activation on a broader level with no significant differences between seropositive and seronegative patients. Taken together, one might assume that UPR-activation within muscle fibers might not only serve to restore protein homeostasis, but also enhance sarcolemmal presentation of proteins crucial for attracting immune cells. Since modulation of ER-stress and UPR via application of chemical chaperones became a promising therapeutic treatment approach, our findings might represent the starting point for new interventional concepts. CA extern

Heme-regulated inhibitor (HRI) kinase is a serine-threonine kinase, controlling the initiation of protein synthesis via phosphorylating α subunit of eIF2 on serine 51 residue, mainly in response to heme deprivation in erythroid cells. However, recent studies showed that HRI is also activated by several diverse signals, causing dysregulations in intracellular homeostatic mechanisms in non-erythroid cells. For instance, it was reported that the decrease in protein synthesis upon the 26S proteasomal inhibition by MG132 or bortezomib is mediated by increased eIF2α phosphorylation in an HRI-dependent manner in mouse embryonic fibroblast cells. The increase in eIF2α phosphorylation level through the activation of HRI upon 26S proteasomal inhibition is believed to protect cells against the buildup of misfolded and ubiquitinated proteins, having the potential to trigger the apoptotic response. In contrast, prolonged and sustained HRI-mediated eIF2α phosphorylation can induce cell death, which may involve ATF4 and CHOP expression. Altogether, these studies suggest that HRI-mediated eIF2α phosphorylation may be cytoprotective or cytotoxic depending on the cells, type, and duration of pharmacological agents used. It is thus hypothesized that both HRI activators, inducing eIF2α phosphorylation or HRI inhibitors causing disturbances in eIF2α phosphorylation, may be effective as novel strategies in cancer treatment if the balance in eIF2α phosphorylation is shifted in favor of autophagic or apoptotic response in cancer cells. It is here aimed to review the role of HRI in various biological mechanisms as well as the therapeutic potentials of recently developed HRI activators and inhibitors, targeting eIF2α phosphorylation in cancer cells.

HIV-1-associated neurocognitive disorders (HAND) are a major concern in HIV-infected individuals despite the currently available antiretroviral therapy regime. Impaired M1 pro-inflammatory microglial activation is considered one of the hallmark features of HAND neuropathogenesis, and it has been suggested that circulant HIV-1 transactivator protein (Tat) can play a critical role in this process. At the same time, endoplasmatic reticulum (ER) stress has also been implicated in neurodegenerative conditions resulting from the accumulation of misfolded proteins and subsequent unfolded protein response (UPR) deflagration. Here, we demonstrate that pharmacological inhibition of UPR-related protein kinase R-like endoplasmic reticulum kinase (PERK) can attenuate HIV-1 Tat-induced M1 inflammatory state in microglia in vitro. Our initial experiments demonstrate that the bystander stimulus of recombinant Tat on BV-2 microglial cells result in the coupled overexpression of central UPR markers and pro-inflammatory mediators such as iNOS, surface CD16/32 and secreted tumour necrosis factor-α (TNF-α), IL-6, monocyte chemoattractant protein (MCP)-1 and NO. We show that blocking PERK-eIF2-α-ATF4 signalling using the PERK inhibitor GSK2606414 leads to reduced inflammatory response in M1-like BV-2 cells activated by recombinant Tat. Taken together, these findings suggest that PERK targeting may provide a therapeutic intervention to mitigate against lasting neuroinflammation and neuronal loss in of HAND.

The present study is designed to determine the effect of LCZ696 on DCM in rats and investigate the underlying mechanism involved. Diabetes was induced by feeding rats with a high-fat diet for six weeks following a single injection of STZ (30 mg/kg). Diabetic rats were divided into three groups (n = 10). LCZ696 and valsartan treatment was started two weeks after diabetic induction and continued for eight weeks. At the end of the treatment, serum and cardiac tissues were analyzed by RT-PCR, Western blot, and ELISA kits. LCZ696 and valsartan ameliorated DCM progression by inhibiting AGEs formation at activity levels; pro-apoptotic markers (BAX/Bcl2 ratio and caspase-3) in mRNA and protein expressions, the NF-κB at mRNA; and protein levels associated with the restoration of elevated proinflammatory cytokines such as the TNF-α, IL-6, and IL-1β at the activity level. Furthermore, LCZ696 and valsartan contribute to restoring the induction of ER stress parameters (GRP78, PERK, eIF2a, ATF4, and CHOP) at mRNA and protein levels. LCZ696 and valsartan attenuated DCM by inhibiting the myocardial inflammation, ER stress, and apoptosis through AGEs/NF-κB and PERK/CHOP signaling cascades. Collectively, the present results reveal that LCZ696 had a more protective solid effect against DCM than valsartan.

Enforcing differentiation of cancer stem cells is considered as a potential strategy to sensitize colorectal cancer cells to irradiation and chemotherapy. Activation of the unfolded protein response, due to endoplasmic reticulum (ER) stress, causes rapid stem cell differentiation in normal intestinal and colon cancer cells. We previously found that stem cell differentiation was mediated by a Protein kinase R-like ER kinase (PERK) dependent arrest of mRNA translation, resulting in rapid protein depletion of WNT-dependent transcription factor c-MYC. We hypothesize that ER stress dependent stem cell differentiation may rely on the depletion of additional transcriptional regulators with a short protein half-life that are rapidly depleted due to a PERK-dependent translational pause. Using a novel screening method, we identify novel transcription factors that regulate the intestinal stem cell fate upon ER stress. ER stress was induced in LS174T cells with thapsigargin or subtilase cytotoxin (SubAB) and immediate alterations in nuclear transcription factor activity were assessed by the CatTFRE assay in which transcription factors present in nuclear lysate are bound to plasmid DNA, co-extracted and quantified using mass-spectrometry. The role of altered activity of transcription factor CtBP2 was further examined by modification of its expression levels using CAG-rtTA3-CtBP2 overexpression in small intestinal organoids, shCtBP2 knockdown in LS174T cells, and familial adenomatous polyposis patient-derived organoids. CtBP2 overexpression organoids were challenged by ER stress and ionizing irradiation. We identified a unique set of transcription factors with altered activation upon ER stress. Gene ontology analysis showed that transcription factors with diminished binding were involved in cellular differentiation processes. ER stress decreased CtBP2 protein expression in mouse small intestine. ER stress induced loss of CtBP2 expression which was rescued by inhibition of PERK signaling. CtBP2 was overexpressed in mouse and human colorectal adenomas. Inducible CtBP2 overexpression in organoids conferred higher clonogenic potential, resilience to irradiation-induced damage and a partial rescue of ER stress-induced loss of stemness. Using an unbiased proteomics approach, we identified a unique set of transcription factors for which DNA-binding activity is lost directly upon ER stress. We continued investigating the function of co-regulator CtBP2, and show that CtBP2 mediates ER stress-induced loss of stemness which supports the intestinal stem cell state in homeostatic stem cells and colorectal cancer cells.