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1.  Endoplasmic Reticulum Stress Response Mediated by the PERK-eIF2α-ATF4 Pathway Is Involved in Osteoblast Differentiation Induced by BMP2* 
The Journal of Biological Chemistry  2010;286(6):4809-4818.
To avoid excess accumulation of unfolded proteins in the endoplasmic reticulum (ER), eukaryotic cells have signaling pathways from the ER to the cytosol or nucleus. These processes are collectively termed the ER stress response. Double stranded RNA activated protein kinase (PKR)-like endoplasmic reticulum kinase (PERK) is a major transducer of the ER stress response and directly phosphorylates eIF2α, resulting in translational attenuation. Phosphorylated eIF2α specifically promotes the translation of the transcription factor ATF4. ATF4 plays important roles in osteoblast differentiation and bone formation. Perk−/− mice are reported to exhibit severe osteopenia, and the phenotypes observed in bone tissues are very similar to those of Atf4−/− mice. However, the involvement of the PERK-eIF2α-ATF4 signaling pathway in osteogenesis is unclear. Phosphorylated eIF2α and ATF4 protein levels were attenuated in Perk−/− calvariae, and the gene expression levels of osteocalcin (Ocn) and bone sialoprotein (Bsp), which are targets for ATF4, were also down-regulated. Treatment of wild-type primary osteoblasts with BMP2, which is required for osteoblast differentiation, induced ER stress, leading to an increase in ATF4 protein expression levels. In contrast, the level of ATF4 in Perk−/− osteoblasts was severely diminished. The results indicate that PERK signaling is required for ATF4 activation during osteoblast differentiation. Perk−/− osteoblasts exhibited decreased alkaline phosphatase activities and delayed mineralized nodule formation relative to wild-type cultures. These abnormalities were almost completely restored by the introduction of ATF4 into Perk−/− osteoblasts. Taken together, ER stress occurs during osteoblast differentiation and activates the PERK-eIF2α-ATF4 signaling pathway followed by the promotion of gene expression essential for osteogenesis, such as Ocn and Bsp.
PMCID: PMC3039352  PMID: 21135100
Bone; Differentiation; ER Stress; Transcription Factors; Transcription Target Genes; Osteoblast
2.  PERK mediates eIF2α phosphorylation responsible for BACE1 elevation, CREB dysfunction and neurodegeneration in a mouse model of Alzheimer’s disease 
Neurobiology of aging  2014;35(10):2272-2281.
Emerging evidence suggests that aberrant phosphorylation of eukaryotic initiation factor-2α (eIF2α) may induce synaptic failure and neurodegeneration through persistent translational inhibition of global protein synthesis. However, elevated phospho-eIF2α also paradoxically causes translational activation of a subset of mRNAs such as the β-secretase enzyme BACE1 and CREB repressor ATF4. Therefore, we tested whether genetic reduction of the eIF2α kinase PERK may prevent these deleterious events and mitigate Alzheimer’s disease (AD)-like neuropathology and cognitive impairments in the 5XFAD mouse model. PERK haploinsufficiency blocked overactivation of the PERK-eIF2α pathway, as evidenced by significant reductions in phosphorylation of PERK and eIF2α, in 5XFAD mice. PERK haploinsufficiency was sufficient to rescue memory deficits and cholinergic neurodegeneration in this AD model. Notably, PERK haploinsufficiency also prevented BACE1 elevations, resulting in reduced levels of amyloid-β peptides and plaque burden in 5XFAD mice. Moreover, CREB dysfunction was restored in PERK+/−·5XFAD mice concomitant with reversal of ATF4 upregulation. Together, these findings suggest that PERK may be a disease-modifying therapeutic target to prevent multiple memory-disrupting mechanisms associated with AD.
PMCID: PMC4127890  PMID: 24889041
PERK; elF2α; BACE1; amyloid-β; ATF4; CREB; cholinergic neuron; fear conditioning; learning and memory; 5XFAD
3.  The eIF2 kinase PERK and the integrated stress response facilitate activation of ATF6 during endoplasmic reticulum stress 
Molecular Biology of the Cell  2011;22(22):4390-4405.
This study shows that the eIF2 kinase PERK is required not only for translational control but also for activation of ATF6 and its target genes in the unfolded protein response. The PERK pathway facilitates both the synthesis of ATF6 and trafficking of ATF6 from the endoplasmic reticulum to the Golgi for intramembrane proteolysis and activation of ATF6.
Disruptions of the endoplasmic reticulum (ER) that perturb protein folding cause ER stress and elicit an unfolded protein response (UPR) that involves translational and transcriptional changes in gene expression aimed at expanding the ER processing capacity and alleviating cellular injury. Three ER stress sensors (PERK, ATF6, and IRE1) implement the UPR. PERK phosphorylation of the α subunit of eIF2 during ER stress represses protein synthesis, which prevents further influx of ER client proteins. Phosphorylation of eIF2α (eIF2α∼P) also induces preferential translation of ATF4, a transcription activator of the integrated stress response. In this study we show that the PERK/eIF2α∼P/ATF4 pathway is required not only for translational control, but also for activation of ATF6 and its target genes. The PERK pathway facilitates both the synthesis of ATF6 and trafficking of ATF6 from the ER to the Golgi for intramembrane proteolysis and activation of ATF6. As a consequence, liver-specific depletion of PERK significantly reduces both the translational and transcriptional phases of the UPR, leading to reduced protein chaperone expression, disruptions of lipid metabolism, and enhanced apoptosis. These findings show that the regulatory networks of the UPR are fully integrated and help explain the diverse biological defects associated with loss of PERK.
PMCID: PMC3216664  PMID: 21917591
4.  Deletion of the eIF2α Kinase GCN2 Fails to Rescue the Memory Decline Associated with Alzheimer’s Disease 
PLoS ONE  2013;8(10):e77335.
Emerging evidence suggests that dysregulated translation through phosphorylation of eukaryotic initiation factor-2α (eIF2α) may contribute to Alzheimer’s disease (AD) and related memory impairments. However, the underlying mechanisms remain unclear. Here, we crossed knockout mice for an eIF2α kinase (GCN2: general control nonderepressible-2 kinase) with 5XFAD transgenic mice, and investigated whether GCN2 deletion affects AD-like traits in this model. As observed in AD brains, 5XFAD mice recapitulated significant elevations in the β-secretase enzyme BACE1 and the CREB repressor ATF4 concomitant with a dramatic increase of eIF2α phosphorylation. Contrary to expectation, we found that GCN2−/− and GCN2+/− deficiencies aggravate rather than suppress hippocampal BACE1 and ATF4 elevations in 5XFAD mice, failing to rescue memory deficits as tested by the contextual fear conditioning. The facilitation of these deleterious events resulted in exacerbated β-amyloid accumulation, plaque pathology and CREB dysfunction in 5XFAD mice with GCN2 mutations. Notably, GCN2 deletion caused overactivation of the PKR-endoplasmic reticulum-related kinase (PERK)-dependent eIF2α phosphorylation pathway in 5XFAD mice in the absence of changes in the PKR pathway. Moreover, PERK activation in response to GCN2 deficiency was specific to 5XFAD mice, since phosphorylated PERK levels were equivalent between GCN2−/− and wild-type control mice. Our findings suggest that GCN2 may be an important eIF2α kinase under the physiological condition, whereas blocking the GCN2 pathway under exposure to significant β-amyloidosis rather aggravates eIF2α phosphorylation leading to BACE1 and ATF4 elevations in AD.
PMCID: PMC3795630  PMID: 24146979
5.  PERK Regulates Working Memory and Protein Synthesis-Dependent Memory Flexibility 
PLoS ONE  2016;11(9):e0162766.
PERK (EIF2AK3) is an ER-resident eIF2α kinase required for memory flexibility and metabotropic glutamate receptor-dependent long-term depression, processes known to be dependent on new protein synthesis. Here we investigated PERK’s role in working memory, a cognitive ability that is independent of new protein synthesis, but instead is dependent on cellular Ca2+ dynamics. We found that working memory is impaired in forebrain-specific Perk knockout and pharmacologically PERK-inhibited mice. Moreover, inhibition of PERK in wild-type mice mimics the fear extinction impairment observed in forebrain-specific Perk knockout mice. Our findings reveal a novel role of PERK in cognitive functions and suggest that PERK regulates both Ca2+ -dependent working memory and protein synthesis-dependent memory flexibility.
PMCID: PMC5023101  PMID: 27627766
6.  miRNA-1283 Regulates the PERK/ATF4 Pathway in Vascular Injury by Targeting ATF4 
PLoS ONE  2016;11(8):e0159171.
In our previous study, we found significant differences in the mRNA and microRNA (miRNA) levels among hypertensive patients with different degrees of vascular endothelial cells damage. These differences were closely associated with endoplasmic reticulum stress (ERS)-related proteins. Moreover, compared to the control group, the expression of transcription factor activating factor 4 (ATF4) was also found to be significantly different in the hypertensive patients with different degrees of vascular endothelial cells damage groups. These results were confirmed using gene prediction software, which showed synergistic effects between ATF4 and miR-1283. ATF4 is a key molecule in ERS. Three ERS pathways exist:protein kinase RNA-like ER kinase (PERK), activating transcription factor 6 (ATF6) and inositol-requiring enzyme-1 (IRE-1)-induced apoptosis. The PERK pathway is the most important and also includes the phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) and ATF4. In this report, we studied the regulatory effects of miR-1283 and ATF4 on the PERK-eIF2α-ATF4 signaling pathway using human umbilical vein endothelial cells (HUVECs) and mice.
Methodology/Principal Findings
We verified the relationship between miR-1283 and ATF4 using a luciferase activity assay and observed the regulatory effects of miR-1283 and ATF4 on the PERK-eIF2α-ATF4 signaling pathway in vivo and in vitro.
ATF4 is a target gene of miR-1283, which regulates the PERK-eIF2α-ATF4 signaling pathway by inhibiting ATF4, and it plays a critical role in inducing injury in HUVECs and mouse heart tissue.
PMCID: PMC4990291  PMID: 27537404
7.  Endoplasmic reticulum stress response in spontaneously hypertensive rats is affected by myocardial ischemia reperfusion injury 
Cell apoptosis induced by endoplasmic reticulum (ER) stress appears to be one of the main causes of myocardial necrosis following myocardial ischemia/reperfusion (MI/R). The C/EBP homologous protein (CHOP) pathway is the main pathway through which apoptosis is induced during ER stress. Glucose-regulated protein 78 (GRP78) is an important protein involved in the CHOP pathway. The present study investigated the hypothesis that MI/R activates the CHOP pathway through signaling via a pathway involving PKR-like ER kinase (PERK), α-subunit of eukaryotic initiation factor 2 (eIF2α) and activating transcription factor 2 (ATF2). Immunohistochemical staining of the heart tissues from spontaneously hypersensitive rats indicated that MI/R injury increases CHOP and GPR78 protein expression levels. To further analyze the mechanism by which MI/R injury induces apoptosis by ER stress, the expression levels of five marker proteins involved in the hypothetical PERK-eIF2α-ATF2 pathway were detected, namely PERK, phosphorylated PERK (P-PERK), eIF2α, phosphorylated eIF2α (P-eIF2α) and ATF2. An increase in the collective expression levels of these proteins would indicate that apoptosis was induced by this signaling pathway. In addition, the study also explored whether hypertension affects the signaling pathway of MI/R-induced myocardial apoptosis by treating spontaneously hypertensive rats (SHRs) with captopril (an effective drug used to treat hypertension). Rats treated with captopril experienced a reduction in blood pressure to normal levels, but no marked differences in the expression levels of the tested proteins or in MI/R injury severity compared with those in untreated rats. These results suggest that MI/R activates the CHOP pathway during ER stress by activating the PERK-eIF2α-ATF2 pathway and that hypertension does not affect this signaling pathway.
PMCID: PMC4280955  PMID: 25574192
endoplasmic reticulum; myocardial necrosis; glucose-regulated protein 78; PKR-like endoplasmic reticulum kinase; α-subunit of eukaryotic initiation factor 2; activating transcription factor 2
8.  Pharmacological brake-release of mRNA translation enhances cognitive memory 
eLife  2013;2:e00498.
Phosphorylation of the α-subunit of initiation factor 2 (eIF2) controls protein synthesis by a conserved mechanism. In metazoa, distinct stress conditions activate different eIF2α kinases (PERK, PKR, GCN2, and HRI) that converge on phosphorylating a unique serine in eIF2α. This collection of signaling pathways is termed the ‘integrated stress response’ (ISR). eIF2α phosphorylation diminishes protein synthesis, while allowing preferential translation of some mRNAs. Starting with a cell-based screen for inhibitors of PERK signaling, we identified a small molecule, named ISRIB, that potently (IC50 = 5 nM) reverses the effects of eIF2α phosphorylation. ISRIB reduces the viability of cells subjected to PERK-activation by chronic endoplasmic reticulum stress. eIF2α phosphorylation is implicated in memory consolidation. Remarkably, ISRIB-treated mice display significant enhancement in spatial and fear-associated learning. Thus, memory consolidation is inherently limited by the ISR, and ISRIB releases this brake. As such, ISRIB promises to contribute to our understanding and treatment of cognitive disorders.
eLife digest
The synthesis of proteins is an essential step in many biological processes, including memory, and drugs that inhibit protein synthesis are known to impair memory in rodents. It is thought that the brain needs these proteins to convert short-term memories into long-term memories through a process known as consolidation.
A protein called EIF2α has a key role in the regulation of protein synthesis, and has also been implicated in memory. EIF2α can be activated as a result of being phosphorylated by any of four protein kinases: these are in turn activated by processes that subject cells to stress, such as viral infection, UV light or—in the case of a kinase known as PERK—the accumulation of unfolded proteins in a cellular organelle called the endoplasmic reticulum. Activation of EIF2α downregulates most protein synthesis inside the cell, but upregulates the production of a small number of key regulatory molecules: these changes help cells to cope with whatever stressful event they have just experienced.
To obtain further insight into the cellular stress response, Sidrauski et al. screened a large library of compounds in search of one that inhibits PERK. They identified a molecule—known as ISRIB—which acts downstream of all four protein kinases by reversing the effects of EIF2α phosphorylation. ISRIB is the first molecule shown to have this effect, and thus represents an important tool for investigating the stress response inside cells.
When Sidrauski et al. injected ISRIB into mice, the animals showed improved memory: for example, they learnt to locate a hidden platform in a water maze more rapidly than controls. This suggests that ISRIB could be used to explore the mechanisms that underlie memory consolidation, and possibly even as a memory enhancer. Moreover, given that many tumor cells exploit the cellular stress response to aid their own growth, ISRIB may have potential as a novel chemotherapeutic agent.
PMCID: PMC3667625  PMID: 23741617
eIF2; eIF2B; ATF4; integrated stress response; unfolded protein response; memory consolidation; Human; Mouse; Rat
9.  PERK regulates Gq protein-coupled intracellular Ca2+ dynamics in primary cortical neurons 
Molecular Brain  2016;9:87.
PERK (EIF2AK3) is an ER-resident eIF2α kinase required for behavioral flexibility and metabotropic glutamate receptor-dependent long-term depression via its translational control. Motivated by the recent discoveries that PERK regulates Ca2+ dynamics in insulin-secreting β-cells underlying glucose-stimulated insulin secretion, and modulates Ca2+ signals-dependent working memory, we explored the role of PERK in regulating Gq protein-coupled Ca2+ dynamics in pyramidal neurons. We found that acute PERK inhibition by the use of a highly specific PERK inhibitor reduced the intracellular Ca2+ rise stimulated by the activation of acetylcholine, metabotropic glutamate and bradykinin-2 receptors in primary cortical neurons. More specifically, acute PERK inhibition increased IP3 receptor mediated ER Ca2+ release, but decreased receptor-operated extracellular Ca2+ influx. Impaired Gq protein-coupled intracellular Ca2+ rise was also observed in genetic Perk knockout neurons. Taken together, our findings reveal a novel role of PERK in neurons, which is eIF2α-independent, and suggest that the impaired working memory in forebrain-specific Perk knockout mice may stem from altered Gq protein-coupled intracellular Ca2+ dynamics in cortical pyramidal neurons.
Electronic supplementary material
The online version of this article (doi:10.1186/s13041-016-0268-5) contains supplementary material, which is available to authorized users.
PMCID: PMC5045583  PMID: 27716400
PERK; Gq protein-coupled receptor; Ca2+; Receptor-operated Ca2+ entry
10.  Transcription factor ATF4 directs basal and stress-induced gene expression in the unfolded protein response and cholesterol metabolism in the liver 
Molecular Biology of the Cell  2016;27(9):1536-1551.
Whole-body and liver-specific ATF4–knockout mice are used to evaluate the role of ATF4 transcription factor in the unfolded protein response (UPR). ATF4 directs a small subset of UPR gene expression, and deletion of ATF4 in the liver triggers enhanced oxidative stress, disrupts cholesterol metabolism, and enhances cell death.
Disturbances in protein folding and membrane compositions in the endoplasmic reticulum (ER) elicit the unfolded protein response (UPR). Each of three UPR sensory proteins—PERK (PEK/EIF2AK3), IRE1, and ATF6—is activated by ER stress. PERK phosphorylation of eIF2 represses global protein synthesis, lowering influx of nascent polypeptides into the stressed ER, coincident with preferential translation of ATF4 (CREB2). In cultured cells, ATF4 induces transcriptional expression of genes directed by the PERK arm of the UPR, including genes involved in amino acid metabolism, resistance to oxidative stress, and the proapoptotic transcription factor CHOP (GADD153/DDIT3). In this study, we characterize whole-body and tissue-specific ATF4-knockout mice and show in liver exposed to ER stress that ATF4 is not required for CHOP expression, but instead ATF6 is a primary inducer. RNA-Seq analysis indicates that ATF4 is responsible for a small portion of the PERK-dependent UPR genes and reveals a requirement for expression of ATF4 for expression of genes involved in oxidative stress response basally and cholesterol metabolism both basally and under stress. Consistent with this pattern of gene expression, loss of ATF4 resulted in enhanced oxidative damage, and increased free cholesterol in liver under stress accompanied by lowered cholesterol in sera.
PMCID: PMC4850040  PMID: 26960794
11.  TBL2 Is a Novel PERK-Binding Protein that Modulates Stress-Signaling and Cell Survival during Endoplasmic Reticulum Stress 
PLoS ONE  2014;9(11):e112761.
Under ER stress, PKR-like ER-resident kinase (PERK) phosphorylates translation initiation factor eIF2α, resulting in repression of global protein synthesis and concomitant upregulation of the translation of specific mRNAs such as activating transcription factor 4 (ATF4). This PERK function is important for cell survival under ER stress and poor nutrient conditions. However, mechanisms of the PERK signaling pathway are not thoroughly understood. Here we identify transducin (beta)-like 2 (TBL2) as a novel PERK-binding protein. We found that TBL2 is an ER-localized type-I transmembrane protein and preferentially binds to the phosphorylated form of PERK, but not another eIF2α kinase GCN2 or ER-resident kinase IRE1, under ER stress. Immunoprecipitation analysis using various deletion mutants revealed that TBL2 interacts with PERK via the N-terminus proximal region and also associates with eIF2α via the WD40 domain. In addition, TBL2 knockdown can lead to impaired ATF4 induction under ER stress or poor nutrient conditions such as glucose and oxygen deprivation. Consistently, TBL2 knockdown rendered cells vulnerable to stresses similarly to PERK knockdown. Thus, TBL2 serves as a potential regulator of the PERK pathway.
PMCID: PMC4231078  PMID: 25393282
12.  Unfolded Protein Response and PERK Kinase as a New Therapeutic Target in the Pathogenesis of Alzheimer’s Disease 
Current medicinal chemistry  2015;22(27):3169-3184.
Recent evidence suggests that the development of Alzheimer’s disease (AD) and related cognitive loss is due to mutations in the Amyloid Precursor Protein (APP) gene on chromosome 21 and increased activation of eukaryotic translation initiation factor-2α (eIF2α) phosphorylation. The high level of misfolded and unfolded proteins loading in Endoplasmic Reticulum (ER) lumen triggers ER stress and as a result Unfolded Protein Response (UPR) pathways are activated. Stress-dependent activation of the protein kinase RNA-like endoplasmic reticulum kinase (PERK) leads to the significant elevation of phospho-eIF2α. That attenuates general translation and, on the other hand, promotes the preferential synthesis of Activating Transcription Factor 4 (ATF4) and secretase β (BACE1) - a pivotal enzyme responsible for the initiation of the amyloidogenic pathway resulting in the generation of the amyloid β (Aβ) variant with high ability to form toxic senile plaques in AD brains. Moreover, excessive, long-term stress conditions may contribute to inducing neuronal death by apoptosis as a result of the overactivated expression of pro-apoptotic proteins via ATF4. These findings allow to infer that dysregulated translation, increased expression of BACE1 and ATF4, as a result of eIF2α phosphorylation, may be a major contributor to structural and functional neuronal loss resulting in memory impairment. Thus, blocking PERK-dependent eIF2α phosphorylation through specific, small-molecule PERK branch inhibitors seems to be a potential treatment strategy for AD individuals. That may contribute to the restoration of global translation rates and reduction of expression of ATF4 and BACE1. Hence, the treatment strategy can block accelerated β-amyloidogenesis by reduction in APP cleaving via the BACE1-dependent amyloidogenic pathway.
PMCID: PMC5010237  PMID: 26282939
Alzheimer’s disease; amyloid β; APP; BACE1; eIF2α; ER stress; PERK inhibitors; PERK; unfolded protein response
13.  Inhibition of Proliferation by PERK Regulates Mammary Acinar Morphogenesis and Tumor Formation 
PLoS ONE  2007;2(7):e615.
Endoplasmic reticulum (ER) stress signaling can be mediated by the ER kinase PERK, which phosphorylates its substrate eIF2α. This in turn, results in translational repression and the activation of downstream programs that can limit cell growth through cell cycle arrest and/or apoptosis. These responses can also be initiated by perturbations in cell adhesion. Thus, we hypothesized that adhesion-dependent regulation of PERK signaling might determine cell fate. We tested this hypothesis in a model of mammary acini development, a morphogenetic process regulated in part by adhesion signaling. Here we report a novel role for PERK in limiting MCF10A mammary epithelial cell proliferation during acinar morphogenesis in 3D Matrigel culture as well as in preventing mammary tumor formation in vivo. We show that loss of adhesion to a suitable substratum induces PERK-dependent phosphorylation of eIF2α and selective upregulation of ATF4 and GADD153. Further, inhibition of endogenous PERK signaling during acinar morphogenesis, using two dominant-negative PERK mutants (PERK-ΔC or PERK-K618A), does not affect apoptosis but results instead in hyper-proliferative and enlarged lumen-filled acini, devoid of proper architecture. This phenotype correlated with an adhesion-dependent increase in translation initiation, Ki67 staining and upregulation of Laminin-5, ErbB1 and ErbB2 expression. More importantly, the MCF10A cells expressing PERKΔC, but not a vector control, were tumorigenic in vivo upon orthotopic implantation in denuded mouse mammary fat pads. Our results reveal that the PERK pathway is responsive to adhesion-regulated signals and that it is essential for proper acinar morphogenesis and in preventing mammary tumor formation. The possibility that deficiencies in PERK signaling could lead to hyperproliferation of the mammary epithelium and increase the likelihood of tumor formation, is of significance to the understanding of breast cancer.
PMCID: PMC1910610  PMID: 17637831
14.  Resistance to Vesicular Stomatitis Virus Infection Requires a Functional Cross Talk between the Eukaryotic Translation Initiation Factor 2α Kinases PERK and PKR 
Journal of Virology  2004;78(23):12747-12761.
Phosphorylation of the alpha (α) subunit of the eukaryotic translation initiation factor 2 (eIF2) leads to the inhibition of protein synthesis in response to diverse stress conditions, including viral infection. The eIF2α kinase PKR has been shown to play an essential role against vesicular stomatitis virus (VSV) infection. We demonstrate here that another eIF2α kinase, the endoplasmic reticulum-resident protein kinase PERK, contributes to cellular resistance to VSV infection. We demonstrate that mouse embryonic fibroblasts (MEFs) from PERK−/− mice are more susceptible to VSV-mediated apoptosis than PERK+/+ MEFs. The higher replication capacity of VSV in PERK−/− MEFs results from their inability to attenuate viral protein synthesis due to an impaired eIF2α phosphorylation. We also show that VSV-infected PERK−/− MEFs are unable to fully activate PKR, suggesting a cross talk between the two eIF2α kinases in virus-infected cells. These findings further implicate PERK in virus infection, and provide evidence that the antiviral and antiapoptotic roles of PERK are mediated, at least in part, via the activation of PKR.
PMCID: PMC524969  PMID: 15542627
15.  PERK‐eIF2α‐ATF4‐CHOP Signaling Contributes to TNFα‐Induced Vascular Calcification 
Vascular calcification is a common feature in patients with chronic kidney disease (CKD). CKD increases serum levels of tumor necrosis factor‐α (TNFα), a critical mediator of vascular calcification. However, the molecular mechanism by which TNFα promotes CKD‐dependent vascular calcification remains obscure. The purpose of the present study was to investigate whether TNFα‐induced vascular calcification in CKD is caused by the endoplasmic reticulum response involving protein kinase RNA‐like endoplasmic reticulum kinase (PERK), eukaryotic initiation factor 2α (eIF2α), activating transcription factor 4 (ATF4), and C/EBP homologous protein (CHOP).
Methods and Results
We examined the effects of TNFα on the endoplasmic reticulum (ER) stress response of vascular smooth muscle cells (VSMCs). TNFα treatment drastically induced the PERK‐eIF2α‐ATF4‐CHOP axis of the ER stress response in VSMCs. PERK, ATF4, and CHOP shRNA‐mediated knockdowns drastically inhibited mineralization and osteogenesis of VSMCs induced by TNFα. CKD induced by 5/6 nephrectomies activated the PERK‐eIF2α‐ATF4‐CHOP axis of the ER stress response in the aortas of ApoE−/− mice with increased aortic TNFα expression and vascular calcification. Treatment of 5/6 nephrectomized ApoE−/− mice with the TNFα neutralizing antibody or chemical Chaperones reduced aortic PERK‐eIF2α‐ATF4‐CHOP signaling of the ER stress increased by CKD. This resulted in the inhibition of CKD‐dependent vascular calcification.
These results suggest that TNFα induces the PERK‐eIF2α‐ATF4‐CHOP axis of the ER stress response, leading to CKD‐dependent vascular calcification.
PMCID: PMC3835225  PMID: 24008080
ATF4; CHOP; endoplasmic reticulum stress; TNFα; vascular calcification
16.  Translational and posttranslational regulation of XIAP by eIF2α and ATF4 promotes ER stress–induced cell death during the unfolded protein response 
Molecular Biology of the Cell  2014;25(9):1411-1420.
Chronic ER stress down-regulates XIAP by activating the PERK branch of the UPR. PERK attenuates Xiap translation via eIF2α phosphor­ylation. PERK promotes XIAP degradation via ATF4. CHOP induction and XIAP suppression act in parallel to sensitize cells to ER stress–induced apoptosis.
Endoplasmic reticulum (ER) protein misfolding activates the unfolded protein response (UPR) to help cells cope with ER stress. If ER homeostasis is not restored, UPR promotes cell death. The mechanisms of UPR-mediated cell death are poorly understood. The PKR-like endoplasmic reticulum kinase (PERK) arm of the UPR is implicated in ER stress–induced cell death, in part through up-regulation of proapoptotic CCAAT/enhancer binding protein homologous protein (CHOP). Chop−/− cells are partially resistant to ER stress–induced cell death, and CHOP overexpression alone does not induce cell death. These findings suggest that additional mechanisms regulate cell death downstream of PERK. Here we find dramatic suppression of antiapoptosis XIAP proteins in response to chronic ER stress. We find that PERK down-regulates XIAP synthesis through eIF2α and promotes XIAP degradation through ATF4. Of interest, PERK's down-regulation of XIAP occurs independently of CHOP activity. Loss of XIAP leads to increased cell death, whereas XIAP overexpression significantly enhances resistance to ER stress–induced cell death, even in the absence of CHOP. Our findings define a novel signaling circuit between PERK and XIAP that operates in parallel with PERK to CHOP induction to influence cell survival during ER stress. We propose a “two-hit” model of ER stress–induced cell death involving concomitant CHOP up-regulation and XIAP down-regulation both induced by PERK.
PMCID: PMC4004591  PMID: 24623724
17.  Regulation of Subtilase Cytotoxin-Induced Cell Death by an RNA-Dependent Protein Kinase-Like Endoplasmic Reticulum Kinase-Dependent Proteasome Pathway in HeLa Cells 
Infection and Immunity  2012;80(5):1803-1814.
Shiga-toxigenic Escherichia coli (STEC) produces subtilase cytotoxin (SubAB), which cleaves the molecular chaperone BiP in the endoplasmic reticulum (ER), leading to an ER stress response and then activation of apoptotic signaling pathways. Here, we show that an early event in SubAB-induced apoptosis in HeLa cells is mediated by RNA-dependent protein kinase (PKR)-like ER kinase (PERK), not activating transcription factor 6 (ATF6) or inositol-requiring enzyme 1(Ire1), two other ER stress sensors. PERK knockdown suppressed SubAB-induced eIF2α phosphorylation, activating transcription factor 4 (ATF4) expression, caspase activation, and cytotoxicity. Knockdown of eIF2α by small interfering RNA (siRNA) or inhibition of eIF2α dephosphorylation by Sal003 enhanced SubAB-induced caspase activation. Treatment with proteasome inhibitors (i.e., MG132 and lactacystin), but not a general caspase inhibitor (Z-VAD) or a lysosome inhibitor (chloroquine), suppressed SubAB-induced caspase activation and poly(ADP-ribose) polymerase (PARP) cleavage, suggesting that the ubiquitin-proteasome system controls events leading to caspase activation, i.e., Bax/Bak conformational changes, followed by cytochrome c release from mitochondria. Levels of ubiquitinated proteins in HeLa cells were significantly decreased by SubAB treatment. Further, in an early event, some antiapoptotic proteins, which normally turn over rapidly, have their synthesis inhibited, and show enhanced degradation via the proteasome, resulting in apoptosis. In PERK knockdown cells, SubAB-induced loss of ubiquitinated proteins was inhibited. Thus, SubAB-induced ER stress is caused by BiP cleavage, leading to PERK activation, not by accumulation of ubiquitinated proteins, which undergo PERK-dependent degradation via the ubiquitin-proteasome system.
PMCID: PMC3347452  PMID: 22354021
18.  PERK (EIF2AK3) Regulates Proinsulin Trafficking and Quality Control in the Secretory Pathway 
Diabetes  2010;59(8):1937-1947.
Loss-of-function mutations in Perk (EIF2AK3) result in permanent neonatal diabetes in humans (Wolcott-Rallison Syndrome) and mice. Previously, we found that diabetes associated with Perk deficiency resulted from insufficient proliferation of β-cells and from defects in insulin secretion. A substantial fraction of PERK-deficient β-cells display a highly abnormal cellular phenotype characterized by grossly distended endoplasmic reticulum (ER) and retention of proinsulin. We investigated over synthesis, lack of ER-associated degradation (ERAD), and defects in ER to Golgi trafficking as possible causes.
ER functions of PERK were investigated in cell culture and mice in which Perk was impaired or gene dosage modulated. The Ins2+/Akita mutant mice were used as a model system to test the role of PERK in ERAD.
We report that loss of Perk function does not lead to uncontrolled protein synthesis but impaired ER-to-Golgi anterograde trafficking, retrotranslocation from the ER to the cytoplasm, and proteasomal degradation. PERK was also shown to be required to maintain the integrity of the ER and Golgi and processing of ATF6. Moreover, decreasing Perk dosage surprisingly ameliorates the progression of the Akita mutants toward diabetes.
PERK is a positive regulator of ERAD and proteasomal activity. Reducing PERK activity ameliorates the progression of diabetes in the Akita mouse, whereas increasing PERK dosage hastens its progression. We speculate that PERK acts as a metabolic sensor in the insulin-secreting β-cells to modulate the trafficking and quality control of proinsulin in the ER relative to the physiological demands for circulating insulin.
PMCID: PMC2911049  PMID: 20530744
19.  Human Cytomegalovirus Protein pUL38 Induces ATF4 Expression, Inhibits Persistent JNK Phosphorylation, and Suppresses Endoplasmic Reticulum Stress-Induced Cell Death▿  
Journal of Virology  2009;83(8):3463-3474.
The endoplasmic reticulum (ER) is a key organelle involved in sensing and responding to stressful conditions, including those resulting from infection of viruses, such as human cytomegalovirus (HCMV). Three signaling pathways collectively termed the unfolded protein response (UPR) are activated to resolve ER stress, but they will also lead to cell death if the stress cannot be alleviated. HCMV is able to modulate the UPR to promote its infection. The specific viral factors involved in such HCMV-mediated modulation, however, were unknown. We previously showed that HCMV protein pUL38 was required to maintain the viability of infected cells, and it blocked cell death induced by thapsigargin. Here, we report that pUL38 is an HCMV-encoded regulator to modulate the UPR. In infection, pUL38 allowed HCMV to upregulate phosphorylation of PKR-like ER kinase (PERK) and the α subunit of eukaryotic initiation factor 2 (eIF-2α), as well as induce robust accumulation of activating transcriptional factor 4 (ATF4), key components of the PERK pathway. pUL38 also allowed the virus to suppress persistent phosphorylation of c-Jun N-terminal kinase (JNK), which was induced by the inositol-requiring enzyme 1 pathway. In isolation, pUL38 overexpression elevated eIF-2α phosphorylation, induced ATF4 accumulation, limited JNK phosphorylation, and suppressed cell death induced by both thapsigargin and tunicamycin, two drugs that induce ER stress by different mechanisms. Importantly, ATF4 overexpression and JNK inhibition significantly reduced cell death in pUL38-deficient virus infection. Thus, pUL38 targets ATF4 expression and JNK activation, and this activity appears to be critical for protecting cells from ER stress induced by HCMV infection.
PMCID: PMC2663240  PMID: 19193809
20.  Coxsackievirus B3 Infection Activates the Unfolded Protein Response and Induces Apoptosis through Downregulation of p58IPK and Activation of CHOP and SREBP1▿  
Journal of Virology  2010;84(17):8446-8459.
Cardiomyocyte apoptosis is a hallmark of coxsackievirus B3 (CVB3)-induced myocarditis. We used cardiomyocytes and HeLa cells to explore the cellular response to CVB3 infection, with a focus on pathways leading to apoptosis. CVB3 infection triggered endoplasmic reticulum (ER) stress and differentially regulated the three arms of the unfolded protein response (UPR) initiated by the proximal ER stress sensors ATF6a (activating transcription factor 6a), IRE1-XBP1 (X box binding protein 1), and PERK (PKR-like ER protein kinase). Upon CVB3 infection, glucose-regulated protein 78 expression was upregulated, and in turn ATF6a and XBP1 were activated via protein cleavage and mRNA splicing, respectively. UPR activity was further confirmed by the enhanced expression of UPR target genes ERdj4 and EDEM1. Surprisingly, another UPR-associated gene, p58IPK, which often is upregulated during infections with other types of viruses, was downregulated at both mRNA and protein levels after CVB3 infection. These findings were observed similarly for uninfected Tet-On HeLa cells induced to overexpress ATF6a or XBP1. In exploring potential connections between the three UPR pathways, we found that the ATF6a-induced downregulation of p58IPK was associated with the activation of PKR (PERK) and the phosphorylation of eIF2α, suggesting that p58IPK, a negative regulator of PERK and PKR, mediates cross-talk between the ATF6a/IRE1-XBP1 and PERK arms. Finally, we found that CVB3 infection eventually produced the induction of the proapoptoic transcription factor CHOP and the activation of SREBP1 and caspase-12. Taken together, these data suggest that CVB3 infection activates UPR pathways and induces ER stress-mediated apoptosis through the suppression of P58IPK and induction/activation of CHOP, SREBP1, and caspase-12.
PMCID: PMC2918999  PMID: 20554776
21.  ER Stress-Induced eIF2-alpha Phosphorylation Underlies Sensitivity of Striatal Neurons to Pathogenic Huntingtin 
PLoS ONE  2014;9(3):e90803.
A hallmark of Huntington’s disease is the pronounced sensitivity of striatal neurons to polyglutamine-expanded huntingtin expression. Here we show that cultured striatal cells and murine brain striatum have remarkably low levels of phosphorylation of translation initiation factor eIF2α, a stress-induced process that interferes with general protein synthesis and also induces differential translation of pro-apoptotic factors. EIF2α phosphorylation was elevated in a striatal cell line stably expressing pathogenic huntingtin, as well as in brain sections of Huntington’s disease model mice. Pathogenic huntingtin caused endoplasmic reticulum (ER) stress and increased eIF2α phosphorylation by increasing the activity of PKR-like ER-localized eIF2α kinase (PERK). Importantly, striatal neurons exhibited special sensitivity to ER stress-inducing agents, which was potentiated by pathogenic huntingtin. We could strongly reduce huntingtin toxicity by inhibiting PERK. Therefore, alteration of protein homeostasis and eIF2α phosphorylation status by pathogenic huntingtin appears to be an important cause of striatal cell death. A dephosphorylated state of eIF2α has been linked to cognition, which suggests that the effect of pathogenic huntingtin might also be a source of the early cognitive impairment seen in patients.
PMCID: PMC3940916  PMID: 24594939
22.  Evidence for eIF2α phosphorylation-independent effects of GSK2656157, a novel catalytic inhibitor of PERK with clinical implications 
Cell Cycle  2014;13(5):801-806.
The endoplasmic reticulum (ER)-resident protein kinase PERK is a major component of the unfolded protein response (UPR), which promotes the adaptation of cells to various forms of stress. PERK phosphorylates the α subunit of the translation initiation factor eIF2 at serine 51, a modification that plays a key role in the regulation of mRNA translation in stressed cells. Several studies have demonstrated that the PERK-eIF2α phosphorylation pathway maintains insulin biosynthesis and glucose homeostasis, facilitates tumor formation and decreases the efficacy of tumor treatment with chemotherapeutic drugs. Recently, a selective catalytic PERK inhibitor termed GSK2656157 has been developed with anti-tumor properties in mice. Herein, we provide evidence that inhibition of PERK activity by GSK2656157 does not always correlate with inhibition of eIF2α phosphorylation. Also, GSK2656157 does not always mimic the biological effects of the genetic inactivation of PERK. Furthermore, cells treated with GSK2656157 increase eIF2α phosphorylation as a means to compensate for the loss of PERK. Using human tumor cells impaired in eIF2α phosphorylation, we demonstrate that GSK2656157 induces ER stress-mediated death suggesting that the drug acts independent of the inhibition of eIF2α phosphorylation. We conclude that GSK2656157 might be a useful compound to dissect pathways that compensate for the loss of PERK and/or identify PERK pathways that are independent of eIF2α phosphorylation.
PMCID: PMC3979916  PMID: 24401334
translation initiation factor eIF2; PERK/PEK kinase; unfolded protein response; protein phosphorylation; mRNA translation; endoplasmic reticulum stress; tumorigenesis; pharmacological inhibitors
23.  Unfolded Protein Response Signaling and MAP Kinase Pathways Underlie Pathogenesis of Arsenic-induced Cutaneous Inflammation 
Arsenic exposure through drinking water is a major global public health problem and is associated with an enhanced risk of various cancers including skin cancer. In human skin, arsenic induces precancerous melanosis and keratosis, which may progress to basal cell and squamous cell carcinoma. However, the mechanism by which these pathophysiological alterations occur remains elusive. In this study, we showed that sub-chronic arsenic exposure to SKH-1 mice induced unfolded protein response (UPR) signaling regulated by proteins, inositol-requiring enzyme-1 (IRE1), PKR-like endoplasmic reticulum kinase (PERK) and activating transcription factor 6 (ATF6). Arsenic activated all three UPR regulatory proteins in the skin. Arsenic induced IRE1 phosphorylation which resulted in augmented splicing of X-box binding protein 1 (XBP-1) leading to its migration to the nucleus, and also enhanced transcriptional activation of downstream target proteins. Hyperphosphorylation of PERK which induces eukaryotic translation initial factor 2 α (eIF2α) in a phosphorylation-dependent manner enhanced translation of ATF4, in addition to augmenting proteolytic activation of ATF6 in arsenic-treated skin. A similar increase in the expression of CHOP was observed. Enhanced XBP-1s, ATF4 and ATF6 regulated downstream chaperones GRP94 and GRP78. Additionally, arsenic induced inflammation-related p38/MAPKAPK-2 MAPK signaling and alterations in Th-1/Th-2/Th-17 cytokines/chemokines and their receptors. Antioxidant N-acetyl cysteine blocked arsenic-induced reactive oxygen species, with a concomitant attenuation of UPR and MAPK signaling and pro-inflammatory cytokine/chemokine signatures. Our results identify novel pathways involved in the pathogenesis of arsenic-mediated cutaneous inflammation which may also be related to enhanced cancer risk in arsenic exposed cohorts.
PMCID: PMC3232315  PMID: 21911445
arsenic; unfolded protein response; MAPK; inflammation; skin
24.  PERK eIF2 alpha kinase is required to regulate the viability of the exocrine pancreas in mice 
BMC Cell Biology  2007;8:38.
Deficiency of the PERK eIF2α kinase in humans and mice results in postnatal exocrine pancreatic atrophy as well as severe growth and metabolic anomalies in other organs and tissues. To determine if the exocrine pancreatic atrophy is due to a cell-autonomous defect, the Perk gene was specifically ablated in acinar cells of the exocrine pancreas in mice.
We show that expression of PERK in the acinar cells is required to maintain their viability but is not required for normal protein synthesis and secretion. Exocrine pancreatic atrophy in PERK-deficient mice was previously attributed to uncontrolled ER-stress followed by apoptotic cell death based on studies in cultured fibroblasts. However, we have found no evidence for perturbations in the endoplasmic reticulum or ER-stress and show that acinar cells succumb to a non-apoptotic form of cell death, oncosis, which is associated with a pronounced inflammatory response and induction of the pancreatitis stress response genes. We also show that mice carrying a knockout mutation of PERK's downstream target, ATF4, exhibit pancreatic deficiency caused by developmental defects and that mice ablated for ATF4's transcriptional target CHOP have a normal exocrine pancreas.
We conclude that PERK modulates secretory capacity of the exocrine pancreas by regulating cell viability of acinar cells.
PMCID: PMC2072952  PMID: 17727724
25.  PERK and GCN2 Contribute to eIF2α Phosphorylation and Cell Cycle Arrest after Activation of the Unfolded Protein Response Pathway 
Molecular Biology of the Cell  2005;16(12):5493-5501.
Exposure of cells to endoplasmic reticulum (ER) stress leads to activation of PKR-like ER kinase (PERK), eukaryotic translation initiation factor 2α (eIF2α) phosphorylation, repression of cyclin D1 translation, and subsequent cell cycle arrest in G1 phase. However, whether PERK is solely responsible for regulating cyclin D1 accumulation after unfolded protein response pathway (UPR) activation has not been assessed. Herein, we demonstrate that repression of cyclin D1 translation after UPR activation occurs independently of PERK, but it remains dependent on eIF2α phosphorylation. Although phosphorylation of eIF2α in PERK–/– fibroblasts is attenuated in comparison with wild-type fibroblasts, it is not eliminated. The residual eIF2α phosphorylation correlates with the kinetics of cyclin D1 loss, suggesting that another eIF2α kinase functions in the absence of PERK. In cells harboring targeted deletion of both PERK and GCN2, cyclin D1 loss is attenuated, suggesting GCN2 functions as the redundant kinase. Consistent with these results, cyclin D1 translation is also stabilized in cells expressing a nonphosphorylatable allele of eIF2α; in contrast, repression of global protein translation still occurs in these cells, highlighting a high degree of specificity in transcripts targeted for translation inhibition by phosphorylated eIF2α. Our results demonstrate that PERK and GCN2 function to cooperatively regulate eIF2α phosphorylation and cyclin D1 translation after UPR activation.
PMCID: PMC1289396  PMID: 16176978

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