Interferons (IFNs) are a family of multifunctional cytokines that activate transcription of subsets of genes. The gene products induced by IFNs are responsible for IFN antiviral, antiproliferative, and immunomodulatory properties. To obtain a more comprehensive list and a better understanding of the genes regulated by IFNs, we compiled data from many experiments, using two different microarray formats. The combined data sets identified >300 IFN-stimulated genes (ISGs). To provide new insight into IFN-induced cellular phenotypes, we assigned these ISGs to functional categories. The data are accessible on the World Wide Web at http://www.lerner.ccf.org/labs/williams/, including functional categories and individual genes listed in a searchable database. The entries are linked to GenBank and Unigene sequence information and other resources. The goal is to eventually compile a comprehensive list of all ISGs. Recognition of the functions of the ISGs and their specific roles in the biological effects of IFNs is leading to a greater appreciation of the many facets of these intriguing and essential cytokines. This review focuses on the functions of the ISGs identified by analyzing the microarray data and focuses particularly on new insights into the protein kinase RNA-regulated (PRKR) protein, which have been made possible with the availability of PRKR-null mice.

OBJECTIVE

Peripheral blood mononuclear cells (PBMCs) from patients with systemic lupus erythematosus (SLE) have increased expression of genes typically induced by type I interferon (IFN). However, it has been difficult to identify and quantify the factors responsible for activation of the IFN pathway in SLE. To characterize these mediators, we developed an assay that measures the functional effects of plasma or serum components on the gene expression of cultured target cells.

METHODS

WISH epithelial cell line cells were cultured with medium, with recombinant IFNalpha, IFNbeta, or IFNgamma, or with 50% plasma from SLE patients (n = 73), rheumatoid arthritis (RA) patients (n = 19), or healthy donors (n = 30). Real-time quantitative polymerase chain reaction was used to determine WISH cell expression of IFN target genes, including PRKR, IFIT1, IFI44, MX1, and C1orf29 (preferentially induced by IFNalpha) and CXCL9 (Mig) (preferentially induced by IFNgamma).

RESULTS

IFNalpha-regulated genes were induced by SLE plasma samples, but not by most of the RA or healthy control plasma samples. The activity in SLE plasma was inhibited >90% by anti-IFNalpha antibody, but not by anti-IFNbeta or anti-IFNgamma antibodies. The expression of each IFNalpha target gene induced by SLE plasma correlated with the expression of that gene studied ex vivo in PBMCs from the same patients and with the titer of anti-RNA binding protein (anti-RBP)-specific autoantibodies. Plasma activity paralleled PBMC expression of IFNalpha-inducible genes over time.

CONCLUSIONS

IFNalpha in SLE plasma is a major stimulus of IFN target gene expression and is related to expression of those genes in PBMCs from SLE patients and to the titer of anti-RBP autoantibodies. These data provide additional support for the view that IFNalpha mediates immune system activation and dysregulation in SLE.

Primary congenital glaucoma (GLC3) is an inherited eye disorder that accounts for 0.01-0.04% of total blindness. Although a large number of chromosomal abnormalities have already been reported in patients with congenital glaucoma, the precise location and pathogenesis of this condition remain elusive. By using a group of 17 GLC3 families and a combination of both candidate regional and general positional mapping strategies, we have mapped a locus for GLC3 to the short arm of chromosome 2. Eleven families showed no recombination with 3 tightly linked markers of D2S177 (Z = 9.40), D2S1346 (Z = 8.83), and D2S1348 (Z = 8.90) with a combined haplotype lod score of 11.50. Haplotype and multipoint linkage analyses of 14 DNA markers from 2p indicated that the disease gene is located in the 2p21 region and is flanked by DNA markers D2S1788/D2S1325 (theta = 0.03; Z = 5.42) and D2S1356 (theta = 0.05; Z = 4.69). Inspection of haplotype and heterogeneity analysis confirmed that 6 families are not linked to the 2p21 region, thus providing the first proof of genetic heterogeneity for this phenotype. We therefore designated the locus on 2p21 GLC3A and positioned it in the overall linkage map of Tel-D2S405-D2S367-(D2S1788/D2S1325)-[(GLC3A++ +, D2S177)/(D2S1346/D2S1348)]-D2S1356-D2S119- D2S1761-D2S1248-D2S1352-D2S406- D2S441-Cen. Of the seven genes mapping to the 2p21 region, CAD, CALM2, and LHCGR are centromeric to D2S119 and can be excluded as a candidate for GLC3A, but mutations in PRKR, TIK, SOS1, or SPTBN1 may still be accountable for this phenotype. As human 2p21 shows homology with mouse chromosomes 11 and 17, the homolog of GLC3A is expected to reside on one of these two chromosomes.

Many etiologies of fatty liver disease (FLD) are associated with the hyperactivation of one of the three pathways composing the unfolded protein response (UPR), which is a harbinger of endoplasmic reticulum (ER) stress. The UPR is mediated by pathways initiated by PRKR-like endoplasmic reticulum kinase, inositol-requiring 1A/X box binding protein 1, and activating transcription factor 6 (ATF6), and each of these pathways has been implicated to have a protective or pathological role in FLD. We used zebrafish with FLD and hepatic ER stress to explore the relationship between Atf6 and steatosis. A mutation of the foie gras (foigr) gene caused FLD and hepatic ER stress. The prolonged treatment of wild-type larvae with tunicamycin (TN), which caused chronic ER stress, phenocopied foigr. In contrast, acute exposure to a high dose of TN robustly activated the UPR but was less effective at inducing steatosis. The sterol regulatory element binding protein transcription factors were not required for steatosis in any of these models. Instead, depleting larvae of active Atf6 either through a membrane-bound transcription factor peptidase site 1 mutation or an atf6 morpholino injection protected them against steatosis caused by chronic ER stress, but exacerbated steatosis caused by acute TN treatment.

CONCLUSIONS

ER stress causes FLD. A loss of Atf6 prevents steatosis caused by chronic ER stress but can also potentiate steatosis caused by acute ER stress. This demonstrates that Atf6 can play both protective and pathological roles in FLD.

Ubiquitous expression of amyotrophic lateral sclerosis (ALS)-causing mutations in superoxide dismutase 1 (SOD1) provokes noncell autonomous paralytic disease. By combining ribosome affinity purification and high-throughput sequencing, a cascade of mutant SOD1-dependent, cell type-specific changes are now identified. Initial mutant-dependent damage is restricted to motor neurons and includes synapse and metabolic abnormalities, endoplasmic reticulum (ER) stress, and selective activation of the PRKR-like ER kinase (PERK) arm of the unfolded protein response. PERK activation correlates with what we identify as a naturally low level of ER chaperones in motor neurons. Early changes in astrocytes occur in genes that are involved in inflammation and metabolism and are targets of the peroxisome proliferator-activated receptor and liver X receptor transcription factors. Dysregulation of myelination and lipid signaling pathways and activation of ETS transcription factors occur in oligodendrocytes only after disease initiation. Thus, pathogenesis involves a temporal cascade of cell type-selective damage initiating in motor neurons, with subsequent damage within glia driving disease propagation.

RET/PTC rearrangements represent key genetic events involved in papillary thyroid carcinoma (PTC) initiation. The aim of the present study was to identify the early changes in gene expression induced by RET/PTC in thyroid cells. For this purpose, microarray analysis was conducted on PCCL3 cells conditionally expressing the RET/PTC3 oncogene. Gene expression profiling 48 h after activation of RET/PTC3 identified a statistically significant modification of expression of 270 genes. Quantitative PCR confirmation of 20 of these demonstrated 90% accuracy of the microarray. Functional clustering of genes with greater than or less than 1.75-fold expression change (86 genes) revealed RET/PTC3-induced regulation of genes with key functions in apoptosis (Ripk3, Tdga), cell-cell signaling (Cdh6, Fn1), cell cycle (Il24), immune and inflammation response (Cxcl10, Scya2, Il6, Gbp2, Oas1, Tap1, RT1Aw2, C2ta, Irf1, Lmp2, Psme2, Prkr), metabolism (Aldob, Ptges, Nd2, Gss, Gstt1), signal transduction (Socs3, Nf1, Jak2, Cpg21, Dusp6, Socs1, Stat1, Stat3, Cish) and transcription (Nr4a1, Junb, Hfh1, Runx1, Foxe1). Genes coding for proteins involved in the immune response and in intracellular signal transduction pathways activated by cytokines and chemokines were strongly represented, indicating a critical role of RET/PTC3 in the early modulation of the immune response.

To characterize the roles of Porphyromonas gingivalis and its components in disease processes, we investigated the cytokine profiles induced by live P. gingivalis, its lipopolysaccharide (LPS), and its major fimbrial protein, fimbrillin (FimA). A cytokine antibody array revealed that human monocyte-derived macrophages were induced to produce chemokines (e.g., monocyte chemoattractant protein 1, macrophage inflammatory protein 1beta [MIP-1beta], and MIP-3alpha) as early as 1 h after exposure to P. gingivalis, with production declining after 24 h of exposure. As expected, an extensive repertoire of inflammatory mediators increased subsequent to infection, most predominantly tumor necrosis factor alpha (TNF-alpha), interleukin 1beta (IL-1beta), IL-6, IL-10, and granulocyte-macrophage colony-stimulating factor. The induction of cytokines by P. gingivalis was not triggered simply by bacterial cell surface components, since purified P. gingivalis LPS and FimA induced similar patterns of cytokines, while the pattern of cytokines induced by live P. gingivalis was significantly different, indicating that the host defense system senses live bacteria differently than it does the cell surface components LPS and FimA. To further understand the mechanisms by which live P. gingivalis and its components exert their effects, we used a high-throughput immunoblot screening approach (Becton-Dickinson PowerBlot) to analyze intracellular proteins involved in P. gingivalis infection in human macrophages. Exposure of human macrophages to either live P. gingivalis, its LPS, or its FimA protein led to the up-regulation of 12, 8, and 10 proteins and the down-regulation of 15, 8, and 17 proteins, respectively. The expression of proteins involved in gene transcription (e.g., monocyte enhancer factor 2D [MEF2D], signal transducer and activator of transcription 1 [STAT1], STAT3, STAT6, and IL enhancer binding factors [ILF3]), of protein kinases (e.g., mitogen-activated protein kinase 3 [MAPK3], MAP3K8, double-stranded RNA-activated protein kinase [PRKR], and MAP2K4), and of proteins involved in immune responses (e.g., TNF super family member 6 [TNFSF6] and interferon-induced protein with tetratricopeptide repeat 4 [IFIT4]), apoptosis (e.g., genes associated with retinoid interferon-induced mortality 19 [GRIM19]), and other fundamental cellular processes (e.g., clathrin heavy-chain polypeptide, culreticulin, and Ras-associated protein RAB27A) was found to be modulated differentially by P. gingivalis, LPS, and FimA. These differential changes are interpreted as preferential signal pathway activation in host immune/inflammatory responses to P. gingivalis infection.

OBJECTIVE

Ionising radiation (IR) can evoke a series of biochemical events inside the cell. However, whether IR can directly induce endoplasmic reticulum (ER) stress is not clear. In our previous study, we found that there might be a causative link between IR and ER stress. In this study, we further characterised the type of ER stress induced by IR.

METHODS

Rat intestinal epithelial cells IEC-6 were irradiated at a dose of 10 Gy, and total RNA and proteins were harvested at indicated time points. The mRNA and protein expression of immunoglobulin heavy chain binding protein (BiP) and glucose regulated protein 94 (GRP94) was detected along with proteins associated with ER stress signal pathways.

RESULTS

Our results indicated that IR induced up-regulation of ER stress marker including BiP and GRP94 at protein and mRNA levels in IEC-6 cells. Increased phosphorylation of eukaryotic translation initiation factor 2 (eIF2alpha) and induced mRNA splicing of X-box binding protein 1 (XBP1) suggested that PERK (interferon-induced double-stranded RNA-activated protein kinase (PRKR) -like endoplasmic reticulum kinase) and IRE1 (inositol requirement 1) signal transduction pathways were involved in this kind of ER stress. However, the active form of activating transcription factor 6 (ATF6) did not change significantly in irradiated cells, which suggested that the ATF6 pathway was not involved.

CONCLUSIONS

Thus, we concluded that IR could induce moderate ER stress directly in IEC-6 cells.

Accumulation of excess hepatic lipids contributes to insulin resistance and liver disease associated with endoplasmic reticulum (ER) stress. Exendin-4 is an agonist of the glucagon-like peptide 1 receptor and plays a role in improving insulin resistance and liver disease by increasing silent mating type information regulation 2 homolog (SIRT) 1. However, the effects and mechanism of action of exendin-4 on responses to palmitic acid (PA)-induced ER stress in hepatocytes have not been clearly defined. We investigated whether exendin-4 attenuates PA-induced ER stress via SIRT1 in HepG2 cells. PA treatment induced increased expression of PRKR-like endoplasmic reticulum kinase, inositol-requiring kinase 1α (IRE1α), activating transcription factor 6 (ATF6), and C/EBP homologous protein (CHOP) mRNA. Exendin-4 decreased the expression of P-IRE1α, ATF6, X-box binding protein-1 and CHOP, and increased the expression of SERCA2b. A significant decrease in the hepatic expression of PUMA, BAX, cytochrome c, and cleaved caspase-3 were observed in hepatocytes treated with exendin-4. The TUNEL assay consistently showed that exendin-4 reversed hepatocyte apoptosis induced by treatment with PA. Inhibition of SIRT1 by nicotinamide and siRNA significantly increased the expression of ER stress marker genes in cells treated with both PA and exendin-4. In conclusion, increased SIRT1 by exendin-4 attenuates PA-induced ER stress and mitochondrial dysfunction in hepatocytes.

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

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

Endoplasmic reticulum (ER) stress plays a vital role in mediating ischemic reperfusion damage in brain. In this study, we evaluated whether melatonin inhibits ER stress in cultured neurons exposed to oxygen and glucose deprivation (OGD) and in rats subjected to transient focal cerebral ischemia. Sprague-Dawley rats were treated with melatonin (5 mg/kg) or control at reperfusion onset after transient occlusion of the right middle cerebral artery (MCA) for 90 min. Brain infarction and hemorrhage within infarcts were measured. The expression of ER stress proteins of phosphorylation of PRKR‑like endoplasmic reticulum kinase (p-PERK), phosphorylation of eukaryotic translation initiation factor 2α (p-eIF2α), activating transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP) were detected by western blotting and immunohistochemistry analysis. The terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) method, cleaved caspase-3 and cytochrome c were used to investigate cell apoptosis in OGD-induced cultured neurons. Our results demonstrated that animals treated with melatonin had significantly reduced infarction volumes and individual cortical lesion sizes as well as increased numbers of surviving neurons. Melatonin can significantly modulate protein levels by decreasing both p-PERK and p-eIF2α in the ischemic core and penumbra. Moreover, the expressions of ATF4 and CHOP were restrained in the ischemic core and penumbra, respectively. Furthermore, pretreatment with melatonin at 10-100 µM effectively reduced the levels of p-PERK and p-eIF2α in cultured neurons after OGD injury. Melatonin treatment also effectively decreased neuron apoptosis resulting from OGD-induced neuron injury. These results indicate that melatonin effectively attenuated post-ischemic ER stress after ischemic stroke.

The cold-inducible RNA-binding motif protein 3 (RBM3) is involved in the protection of neurons in hypoxic-ischemic and neurodegenerative disorders. RBM3 belongs to a small group of proteins whose synthesis increases during hypothermia while global protein production is slowed down. To investigate the molecular mechanisms underlying RBM3 action, we subjected hippocampal organotypic slice cultures from RBM3 knockout mice to various stressors and found exuberant signaling of the endoplasmic reticulum (ER) stress pathway PRKR-like ER kinase (PERK)-eukaryotic translation initiation factor 2α (eIF2α)-CCAAT/enhancer-binding protein homologous protein (CHOP) as compared with wild-type mice. Further, blocking RBM3 expression in human embryonic kidney HEK293 cells by specific small interfering RNAs increased phosphorylation of PERK and eIF2α, whereas overexpression of RBM3 prevented PERK-eIF2α-CHOP signaling during ER stress induced by thapsigargin or tunicamycin. RBM3 did not affect expression of the ER stress sensor immunoglobulin binding protein/GRP78. However, based on affinity purification coupled with mass spectrometry, coimmunoprecipitation, and proximity ligation assay, we revealed that nuclear factor 90 (NF90) is a novel protein interactor of PERK and that this interaction is essential for RBM3-mediated regulation of PERK activity, which requires an RNA-dependent interaction. In conclusion, our data provide evidence for a central role of RBM3 in preventing cell death by inhibiting the PERK-eIF2α-CHOP ER stress pathway through cooperation with NF90.

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

Ca(2+) may trigger apoptosis in β-cells. Hence, the control of intracellular Ca(2+) may represent a potential approach to prevent β-cell apoptosis in diabetes. Our objective was to investigate the effect and mechanism of action of plasma membrane Ca(2+)-ATPase (PMCA) overexpression on Ca(2+)-regulated apoptosis in clonal β-cells. Clonal β-cells (BRIN-BD11) were examined for the effect of PMCA overexpression on cytosolic and mitochondrial [Ca(2+)] using a combination of aequorins with different Ca(2+) affinities and on the ER and mitochondrial pathways of apoptosis. β-cell stimulation generated microdomains of high [Ca(2+)] in the cytosol and subcellular heterogeneities in [Ca(2+)] among mitochondria. Overexpression of PMCA decreased [Ca(2+)] in the cytosol, the ER, and the mitochondria and activated the IRE1α-XBP1s but inhibited the PRKR-like ER kinase-eIF2α and the ATF6-BiP pathways of the ER-unfolded protein response. Increased Bax/Bcl-2 expression ratio was observed in PMCA overexpressing β-cells. This was followed by Bax translocation to the mitochondria with subsequent cytochrome c release, opening of the permeability transition pore, and apoptosis. In conclusion, clonal β-cell stimulation generates microdomains of high [Ca(2+)] in the cytosol and subcellular heterogeneities in [Ca(2+)] among mitochondria. PMCA overexpression depletes intracellular [Ca(2+)] stores and, despite a decrease in mitochondrial [Ca(2+)], induces apoptosis through the mitochondrial pathway. These data open the way to new strategies to control cellular Ca(2+) homeostasis that could decrease β-cell apoptosis in diabetes.

The genes encoding P68 and P65 (PRKR), the human and mouse interferon-inducible dsRNA-dependent protein kinases, respectively, have been mapped to a single locus on human chromosome 2 (band p21) and on mouse chromosome 17 (band E2). These kinases have been implicated in the antiviral response mediated by interferon since their activation by virus-specific dsRNAs can lead to the inhibition of protein synthesis. Recently we have shown that the dsRNA-dependent kinase also may function as a tumor suppressor gene since defective mutant proteins induced malignant transformation. Identification of the chromosomal location of human PRKR permitted a survey of translocations, deletions, or other rearrangement events involving this segment of chromosome 2 in a variety of human malignancies. Finally, our results define a new region of conservation between the distal part of the short arm of chromosome 2 (band p21) and band E2 of mouse chromosome 17.

The interferon-induced dsRNA-activated protein kinase (PRKR) belongs to a subclass of serine/threonine kinases, involved in the regulation of protein synthesis by phosphorylation of the alpha subunit of initiation factor eIF2. Somatic cell hybrids segregating human chromosomes were used to assign this kinase to human chromosome 2. Fluorescence in situ hybridization confirmed this assignment and further localized the gene (PRKR) to the boundary region of bands p21 and 22.

The endoplasmic reticulum (ER) is the site of protein folding and secretion, Ca²⁺ storage and lipid synthesis in eukaryotic cells. Disruption to protein folding or Ca²⁺ homeostasis in the ER leads to the accumulation of unfolded proteins, a condition known as ER stress. This leads to activation of the unfolded protein response (UPR) pathway in order to restore protein homeostasis. Three ER membrane proteins, namely inositol-requiring enzyme 1 (IRE1), protein kinase RNA-like ER kinase (PERK) and activating transcription factor 6 (ATF6), sense the accumulation of unfolded/misfolded proteins and are activated, initiating an integrated transcriptional programme. Recent literature demonstrates that activation of these sensors can alter lipid enzymes, thus implicating the UPR in the regulation of lipid metabolism. Given the presence of ER stress and UPR activation in several diseases including cancer and neurodegenerative diseases, as well as the growing recognition of altered lipid metabolism in disease, it is timely to consider the role of the UPR in the regulation of lipid metabolism. This review provides an overview of the current knowledge on the impact of the three arms of the UPR on the synthesis, function and regulation of fatty acids, triglycerides, phospholipids and cholesterol.

Keywords: PRKR-like endoplasmic reticulum kinase; activating transcription factor 6; cholesterol; endoplasmic reticulum; fatty acid; inositol-requiring enzyme 1; lipid metabolism; phospholipid; triglyceride; unfolded protein response.

Endoplasmic reticulum (ER) stress is caused by the disturbance of ER homeostasis and leads to the activation of the unfolded protein response (UPR), which alleviates stress at an early stage and triggers apoptosis if homeostasis fails over a prolonged timeframe. Here, we report that reticulocalbin 1 (RCN1), a member of the CREC family, is transactivated by nuclear factor kappa B (NF-κB) during ER stress and inhibits ER stress-induced apoptosis. The depletion of RCN1 increases the UPR during drug-induced ER stress by activating PRKR-like ER kinase-CCAAT/enhancer-binding protein-homologous protein (PERK-CHOP) signaling, thus inducing apoptosis. Furthermore, we found that the first two EF-hand calcium-binding motifs of RCN1 specifically interact with inositol 1,4,5-trisphosphate (IP3) receptor type 1 (IP3R1) on loop 3 of its ER luminal domain and inhibit ER calcium release and apoptosis. Together, these data indicate that RCN1, a target of NF-κB, suppresses ER calcium release by binding to IP3R1 and decreases the UPR, thereby inhibiting ER stress-induced apoptosis.

Cytosolic and organelle-based heat-shock protein (HSP) chaperones ensure proper folding and function of nascent and injured polypeptides to support cell growth. Under conditions of cellular stress, including oncogenic transformation, proteostasis components maintain homeostasis and prevent apoptosis. Although this cancer-relevant function has provided a rationale for therapeutically targeting proteostasis regulators (e.g., HSP90), cancer-subtype dependencies upon particular proteostasis components are relatively undefined. Here, we show that human rhabdomyosarcoma (RMS) cells, but not several other cancer cell types, depend upon heat-shock protein 70 kDA (HSP70) for survival. HSP70-targeted therapy (but not chemotherapeutic agents) promoted apoptosis in RMS cells by triggering an unfolded protein response (UPR) that induced PRKR-like endoplasmic reticulum kinase (PERK)-eukaryotic translation initiation factor α (eIF2α)-CEBP homologous protein (CHOP) signaling and CHOP-mediated cell death. Intriguingly, inhibition of only cytosolic HSP70 induced the UPR, suggesting that the essential activity of HSP70 in RMS cells lies at the endoplasmic reticulum-cytosol interface. We also found that increased CHOP mRNA in clinical specimens was a biomarker for poor outcomes in chemotherapy-treated RMS patients. The data suggest that, like human epidermal growth factor receptor 2 (HER2) amplification in breast cancer, increased CHOP in RMS is a biomarker of decreased response to chemotherapy but enhanced response to targeted therapy. Our findings identify the cytosolic HSP70-UPR axis as an unexpected regulator of RMS pathogenesis, revealing HSP70-targeted therapy as a promising strategy to engage CHOP-mediated apoptosis and improve RMS treatment. Our study highlights the utility of dissecting cancer subtype-specific dependencies on proteostasis networks to uncover unanticipated cancer vulnerabilities.

A range of adverse, early life environmental influences such as viral infection and social deprivation are thought to increase risk of psychiatric illness later in life. Here, we used peripheral administration of the viral infection mimic polyriboinosinic-polyribocytidylic acid (polyI:C) to compare the consequences of peripubertal infection and isolation rearing. Isolation rearing induced deficits in sensorimotor gating and recognition memory while no changes in social interaction or spatial learning were observed. PolyI:C injection during the peripubertal period markedly increased expression of interferon-stimulated genes (Ifit2, Prkr, Mx2 and Irf7) in the hippocampal dentate gyrus demonstrating that peripheral administration of the viral mimic in the adolescent animal does have direct effects in the brain. Peripubertal infection mimicry induced a similar but later emerging behavioural deficit in prepulse inhibition implying the existence of a peripubertal window of opportunity for viral-mediated cytokine increases to impact brain development and function. PolyI:C treatment also impaired novel object recognition but did not alter spatial reference memory or social interaction. Combining the polyI:C challenge with social isolation did not exacerbate the behavioural deficits seen with isolation rearing alone. Using Irf7 as a marker, peripubertal viral infection mimicry, isolation rearing and a combination of both were all seen to produce a long-lasting molecular imprint on the interferon-associated signalling pathway in the principal neuron population of the hippocampal dentate gyrus. The data suggest that the sensitivity of brain structure and function to disruption by viral infection extends into the peripubertal period. Moreover, augmented interferon signalling in hippocampus may represent a common molecular imprint of environmental insults associated with neuropsychiatric illnesses like schizophrenia.

OBJECTIVE

Hydroxycamptothecin (HCPT) has been proven to induce apoptosis in fibroblasts. In this study, we investigated whether the PRKR-like ER kinase (PERK) pathway is implicated in apoptotic signaling of human Tenon's capsule fibroblasts (HTCFs) by HCPT.

METHODS

Normal and PERK-knockdown HTCFs were used in this study. Apoptosis was determined by the cell viability assay, Annexin V/propidium iodide (PI) dual-staining, cell cycle analysis in HTCFs treated with HCPT in various doses and for various durations. Endoplasmic reticulum (ER) stress markers and sensor proteins were detected by Western blot analysis. Mitochondrial dysfunction was measured by detecting the mitochondrial membrane potential (ΔΨm) and measuring the expression of cytochrome c (cyt c).

RESULTS

HCPT induced apoptosis in the HTCFs, which was characterized as decreased cell viability and sub-S fraction of the cell cycle and increased apoptosis rate by Annexin V/PI dual-staining. The activity levels of caspase-3 and caspase-9 were significantly increased and were accompanied by cytosolic release of cyt c and decreased ΔΨm in response to HCPT. Treatment with HCPT increased the expression of glucose-regulated protein 78 (GRP78), phospho-PERK, activating transcription factor 6 (ATF6), phosphoinositol-requiring kinase 1 (IRE1), C/EBP homologous protein (CHOP), Bax, and phospho-c-Jun N-terminal kinase (JNK) and decreased the expression of Bcl-2. Knockdown of PERK attenuates HCPT-induced apoptosis in HTCFs, dependent upon both ER stress and the mitochondrial apoptotic pathway.

CONCLUSIONS

This study suggests that the ER stress response and mitochondrial dysfunction are involved in apoptosis induced by HCPT in HTCFs, which might be mediated by PERK; thus, this study offers new insight into preventing postoperative scarring via treatment with HCPT.