INTRODUCTION The endoplasmic reticulum (ER) is a site for calcium storage, lipid biosynthesis, and the folding and assembly of proteins destined for the secretory pathway. Calcium dysregulation, oxidative damage, and perturbations in posttranslational modification of proteins can lead to accumulation of misfolded protein that can cause ER stress. Accumulation of misfolded protein and the ensuing ER stress is referred to as proteotoxicity, which can contribute to the etiology of diseases including diabetes, cancer, and neurodegenerative disorders (Schroder and Kaufman, 2005; Marciniak and Ron, 2006; Ron and Walter, 2007; Wek and Cavener, 2007; Hotamisligil, 2010). ER stress activates three transmembrane proteins—ATF6, IRE1 (ERN1), and PERK (PEK/EIF2AK3)—which together induce the unfolded protein response (UPR), involving both transcriptional and translational regulation of genes that serve to expand the processing capacity of the ER and return this organelle to homeostasis (Ron and Walter, 2007; Wek and Cavener, 2007). ATF6 is a membrane-bound transcription factor that is localized to the ER and serves as both a sensor of ER stress and a transcriptional activator of UPR target genes. There are two ER stress-responsive isoforms of ATF6 (α/β) (Haze et al., 2001; Thuerauf et al., 2004, 2007), with the predominant activator of UPR target genes, ATF6α (ATF6), being the primary focus of this study. During ER stress, ATF6 transits from the ER to the Golgi, where regulated intramembrane proteolysis by site-1-protease (S1P) and site-2-protease (S2P) liberates the N-terminal portion of ATF6 (DeBose-Boyd et al., 1999; Shen and Prywes, 2004; Ron and Walter, 2007). The resulting ATF6(N) then enters the nucleus and binds to ER stress response elements and unfolded protein response elements (ERSEs and UPREs) located in the promoters of UPR targeted genes (Yamamoto et al., 2004, 2007; Ron and Walter, 2007; Wu et al., 2007; Adachi et al., 2008). Activation of ATF6 leads to increased transcription of a network of genes, including those encoding ER chaperones, such as BiP/GRP78, and components of the ER-associated degradation (ERAD) pathway (Ron and Walter, 2007; Wu et al., 2007; Yamamoto et al., 2007; Adachi et al., 2008). Deletion of ATF6 in mice does not lead to an overt developmental phenotype; however, ATF6-null mice subjected to pharmacological induction of ER stress showed dysregulation of chaperone and metabolic genes associated with lipid homeostasis leading to hepatic steatosis not found in the similarly treated wild-type (WT) animals (Wu et al., 2007; Yamamoto et al., 2007, 2010; Rutkowski et al., 2008). Central to the UPR dysregulation in the ATF6 −/− mice is a persistent activation of IRE1, an endoribonuclease that facilitates the cytoplasmic splicing of XBP1 mRNA, leading to the synthesis of an active form of the XBP1 transcription factor (Yoshida et al., 2001; Calfon et al., 2002; Schroder and Kaufman, 2005). Activated XBP1 enhances the transcription of UPR genes involved in protein quality control, disulfide linkage, ERAD, and lipid synthesis (Ron and Walter, 2007; Lee et al., 2008; Glimcher, 2010). The endoribonuclease function of IRE1 also facilitates the degradation of many mRNAs during ER stress, further contributing to changes of the transcriptome (Hollien and Weissman, 2006; Hollien et al., 2009). The translational control arm of the UPR is directed by PERK phosphorylation of eIF2, a translation initiator factor that combines with guanosine triphosphate (GTP) and delivers the initiator Met-tRNAi Met to the translational machinery. Phosphorylation of the α subunit of eIF2 at Ser-51 blocks the activity of eIF2B required for the exchange of eIF2-GDP (guanosine diphosphate) to the active version eIF2-GTP (Wek and Cavener, 2007; Sonenberg and Hinnebusch, 2009). The subsequent reduction in protein synthesis prevents further influx of nascent polypeptides into the ER and provides the cell ample time to implement the UPR reprogramming of the transcriptome. Accompanying this repression of global protein synthesis, eIF2α phosphorylation (eIF2α∼P) also enhances the translation of select mRNAs, such as that encoding ATF4, a transcriptional activator of genes involved in metabolism, cellular redox status, and regulation of apoptosis (Harding et al., 2000a, 2003; Lu et al., 2004; Vattem and Wek, 2004; Ron and Walter, 2007; Wek and Cavener, 2007). Among the ATF4 target genes are additional transcription factors, such as CHOP (GADD153/DDIT3), which serve to amplify the restructuring of the transcriptome to manage stress or direct cell fate toward apoptosis (Fawcett et al., 1999; Harding et al., 2000a; Marciniak et al., 2004; Marciniak and Ron, 2006). In addition to ER stress, preferential translation of ATF4 occurs in response to diverse stress conditions that regulate other eIF2α kinases, including GCN2 (EIF2AK4) activated by nutrient deprivation, HRI (EIF2AK1) induced by heme deficiency and oxidative stress, and PKR (EIF2AK2), which participates in the cellular defense against viral infection (Wek and Cavener, 2007; Sonenberg and Hinnebusch, 2009). Because ATF4 is a common downstream target of each of the eIF2α kinases and their corresponding activating stress signals, this eIF2α∼P/ATF4 pathway has been referred to as the integrated stress response (ISR; Harding et al., 2000a, 2003). The ISR network is critical for ameliorating stress conditions, such as those afflicting the ER organelle, as genetic perturbations of the eIF2α kinases have significant medical consequences. For example, PERK disruption leads to Wolcott-Rallison syndrome, which is characterized by neonatal diabetes, atrophy of the exocrine pancreas, skeletal dysplasia, growth retardation, and hepatic complications resulting in morbidity (Delepine et al., 2000; Senee et al., 2004; Julier and Nicolino, 2010). These pathologies were recapitulated in PERK-deficient mice (Harding et al., 2001; Zhang et al., 2002), in which the molecular and cellular mechanisms have been investigated (Li et al., 2003; Zhang et al., 2006b; Iida et al., 2007; Gupta et al., 2010). Activation of ATF6, IRE1, and PERK in response to ER stress is thought to occur in parallel, but the timing or duration of each may differ. Although the mechanistic details are not yet resolved, it has been proposed that the ER luminal portions of each of these sensory proteins can be bound and repressed by BiP (Shen et al., 2002; Ron and Walter, 2007). Accumulation of unfolded proteins in the ER lumen is suggested to compete with the sensory proteins for BiP binding, enhancing release of this ER chaperone from each ER sensor and thus leading to their activation (Bertolotti et al., 2000; Ma et al., 2002; Shen et al., 2002; Ron and Walter, 2007; Pincus et al., 2010). Alternatively, it has been proposed that unfolded proteins can directly interact with the ER sensor proteins, such as IRE1, which then facilitates oligomerization and activation (Credle et al., 2005; Kimata et al., 2007; Ron and Walter, 2007; Cui et al., 2011). These models posit that activation of the sensory proteins in response to ER stress occurs largely by independent rather than by interconnected processes. We wished to determine whether the translational control arm of the UPR is coupled to the regulation of the transcriptional components of this stress response, insuring coordinate control of the transcription and translation phases of the UPR. Our studies show that the PERK/eIF2α∼P/ATF4 pathway is required to facilitate activation of ATF6 in response to ER stress both in vivo and in cultured cells. As a consequence, liver-specific deletion of PERK leads to enhanced apoptosis in response to ER stress, along with hepatic dysfunctions, features that are similar to those described for ATF6-null mice. These findings are important because development of specific therapies targeting the UPR will depend on understanding the integration of this signaling network. In this model, loss of translational control of the UPR also significantly blocks the transcriptional arms, resulting in dysregulation of the UPR network that would ultimately contribute to disease. RESULTS Loss of PERK disrupts the UPR and renders the liver susceptible to ER stress To address the role of PERK in the UPR in liver, we bred mice with a liver-specific knockout of PERK (LsPERK-KO). LsPERK-KO mice were produced by deletion of floxed PERK fl/fl using cre expression driven by the liver-specific albumin promoter, as described previously (Zhang et al., 2002; Bunpo et al., 2009). Consistent with PERK depletion from the liver, there was significantly reduced eIF2α∼P following 6 h after injection with tunicamycin (1 mg/kg), a potent ER stress agent that blocks N-glycosylation of proteins (Figure 1, A and B). Disruption of eIF2α∼P in the LsPERK-KO livers was also observed following 24- and 36-h treatments with tunicamycin. In addition to translational expression of ATF4, induced eIF2α∼P enhances the transcription of ATF4 and its target gene CHOP, and the levels of both mRNAs were sharply reduced in the LsPERK-KO mice during ER stress (Figure 1C). Expression of CREB-H, which has been previously linked to the UPR (Zhang et al., 2006a), was unchanged during the treatment with tunicamycin. FIGURE 1: Liver-specific knockout of PERK reduces eIF2α∼P and the ISR in response to tunicamycin treatment. (A) WT and LsPERK-KO mice received intraperitoneal injections with tunicamycin (TUN), and following 6, 24, or 36 h of exposure to this ER stress agent, the levels of eIF2α∼P and total eIF2α in livers were measured by immunoblot analyses. The + indicates treatment with tunicamycin, and the – indicates injection with only excipient. Results shown in each panel are representative of at least three independent experiments. (B) Quantification of the levels of total and phosphorylated eIF2α in the WT and LsPERK-KO livers following 6 h of treatment with tunicamycin (TUN) or with excipient (NT, no treatment). (C) The levels of ATF4, CHOP, BiP, and CREB-H mRNAs in the WT and LsPERK-KO livers exposed to tunicamycin for 6 h, or to no treatment, were measured by qPCR. * indicates statistically significant differences (p 1.0 ≥2.0 1.0-fold and ≥2.0-fold thresholds. The repressed genes are also shown for the 0.05 or FDR > 0.15), such that the resulting data set would further represent ER stress-dependent genes. From these ER stress-dependent data, probe sets that had an interaction p value (condition * genotype) of p > 0.05 were removed to enrich the subset of PERK-dependent genes. Values of p for each statistical test and FDR values for each comparison are listed in Supplemental Tables 1, 2, and 3. The following gene ontology (GO) terms were used to identify genes in the PERK-dependent data set that were linked with ER-to-Golgi transport: Primary GO term, Biological process: 0006810 (transport). Secondary GO terms, Gene Ontology Cellular Compartment: 0005783 (endoplasmic reticulum), 0005789 (endoplasmic reticulum membrane), 0000139 (Golgi membrane), 0005794 (Golgi apparatus). Microarray data have been deposited in GEO (gene expression omnibus; www.ncbi.nlm.nih.gov/geo) under the accession number GSE29929. Cell culture PERK −/−, ATF4 −/−, and A/A MEF cells, and their WT counterparts, have been described previously (Jiang et al., 2004). We also analyzed ATF4 −/− MEF cells that were derived from an independent knockout (Hettmann et al., 2000), as presented in Supplemental Figure 3. These MEF cells were grown in DMEM (4.5 g/l glucose), supplemented with 1X MEM nonessential amino acids (HyClone SH30238.01), 1X MEM essential amino acids (HyClone SH30598.01), and 50 μM β-mercaptoethanol, as described previously (Harding et al., 2003). Cultured cells were treated with 2 μM tunicamycin, 1 μM thapsigargin, 1 μM MG132, 10 μM actinomycin D, 50 μg/ml cycloheximide, 5 μg/ml brefeldin A, and 10 μM Sal 003 as indicated. Specific experimental details regarding pretreatments or cotreatments are provided in figure legends when more than one drug was used. Luciferase assays Luciferase assays were carried out in six-well plates using the Dual-Luciferase reporter assay system (Promega, Madison, WI) following the manufacturer's instructions. The ATF6 expression plasmids were described by Prywes and colleagues (Wang et al., 2000). For measurements of ATF6 transcriptional activity, plasmid p912 encoding five ATF6-binding elements fused to the firefly luciferase reporter gene was cotransfected with a Renilla normalization plasmid into PERK −/−, ATF4 −/−, A/A, and WT MEF cells. Measurements were determined as the relative light units (RLU) of the firefly luciferase normalized to Renilla luciferase. Plasmid p1018 expressing ATF6 encoding residues 1-500Δ431-475 (Shen et al., 2002) was cotransfected with p912 and the Renilla normalization plasmid into WT and ATF4 −/− MEF cells, and the ATF6 transcriptional activity with the cotransfection of ATF6-1-500Δ431-475 was determined. The activity of the ATF6 gene promoter was measured by using a luciferase reporter assay. A DNA segment encoding −1500 to −1 base pairs relative to the transcriptional start site of the ATF6 gene was amplified using the primers F5′-GGGACGCGTGCAAACGTGCAGCCTGGTCTGTATGTGGGTCCCC-3′ and R5′-GGGCTCGAGCACCGCCCCGTGGCCTCCTGCCGCGCCCAGCCTTTCTAGG-3′, with the Mlu1 and Xho1 restriction sites underlined. The resulting PCR product was then digested, and the DNA fragment was inserted using the Mlu1 and Xho1 restriction sites of the pGL3-basic vector (Promega), resulting in p1052. The plasmid p1052 was cotransfected with the Renilla vector into ATF4 −/−, and WT MEF cells and the ATF6 promoter activity was determined using the dual luciferase assay. Statistics Data were analyzed using two-way ANOVA to assess main and interaction effects, with drug and genotype as the independent variables (StatSoft, Tulsa, OK). When a significant overall effect was detected, differences among treatment groups were assessed with Duncan's multiple range post hoc test. The level of significance was set at p < 0.05 for all statistical tests. Supplementary Material Supplemental Materials
