Title: Herp Is Dually Regulated by Both the Endoplasmic Reticulum Stress-specific Branch of the Unfolded Protein Response and a Branch That Is Shared with Other Cellular Stress Pathways
Abstract: The mammalian unfolded protein response (UPR) includes two major branches: one(s) specific to ER stress (Ire1/XBP-1 and ATF6-dependent), and one(s) shared by other cellular stresses (PERK/eIF-2α phosphorylation-dependent). Here, we demonstrate that the ER-localized protein Herp represents a second target, in addition to CHOP, that is dually regulated by both the shared and the ER stress-specific branches during UPR activation. For the first time, we are able to assess the contribution of each branch of the UPR in the induction of these targets. We demonstrate that activation of the shared branch of the UPR alone was sufficient to induce Herp and CHOP. ATF4 was not required during ER stress when both branches were used but did contribute significantly to their induction. Conversely, stresses that activated only the shared branch of the UPR were completely dependent on ATF4 for CHOP and Herp induction. Thus, the shared and the ER stress-specific branches of the UPR diverge to regulate two groups of targets, one that is ATF6 and Ire1/XBP-1-dependent, which includes BiP and XBP-1, and another that is eIF-2α kinase-dependent, which includes ATF4 and GADD34. The two branches also converge to maximally up-regulate targets like Herp and CHOP. Finally, our studies reveal that a PERK-dependent target other than ATF4 is contributing to the cross-talk between the two branches of the UPR that has previously been demonstrated. The mammalian unfolded protein response (UPR) includes two major branches: one(s) specific to ER stress (Ire1/XBP-1 and ATF6-dependent), and one(s) shared by other cellular stresses (PERK/eIF-2α phosphorylation-dependent). Here, we demonstrate that the ER-localized protein Herp represents a second target, in addition to CHOP, that is dually regulated by both the shared and the ER stress-specific branches during UPR activation. For the first time, we are able to assess the contribution of each branch of the UPR in the induction of these targets. We demonstrate that activation of the shared branch of the UPR alone was sufficient to induce Herp and CHOP. ATF4 was not required during ER stress when both branches were used but did contribute significantly to their induction. Conversely, stresses that activated only the shared branch of the UPR were completely dependent on ATF4 for CHOP and Herp induction. Thus, the shared and the ER stress-specific branches of the UPR diverge to regulate two groups of targets, one that is ATF6 and Ire1/XBP-1-dependent, which includes BiP and XBP-1, and another that is eIF-2α kinase-dependent, which includes ATF4 and GADD34. The two branches also converge to maximally up-regulate targets like Herp and CHOP. Finally, our studies reveal that a PERK-dependent target other than ATF4 is contributing to the cross-talk between the two branches of the UPR that has previously been demonstrated. The accumulation of malfolded proteins in the endoplasmic reticulum (ER) 1The abbreviations used are: ER, endoplasmic reticulum; CAT, chloramphenicol acetyltransferase; CHOP, C/EBP homologous protein; EMSA, electrophoretic mobility shift assay; ERSE, ER stress response element; UPR, unfolded protein response; MEF, mouse embryonic fibroblasts; eIF, eukaryotic initiation factor; PERK, PKR-like ER kinase. activates a cytoprotective signaling cascade termed the unfolded protein response (UPR). Proximal transducers of the mammalian UPR, which include PERK, Ire1, and ATF6, are all ER-localized transmembrane proteins (1Harding H.P. Zhang Y. Ron D. Nature. 1999; 397: 271-274Google Scholar, 2Shi Y. Vattem K.M. Sood R. An J. Liang J. Stramm L. Wek R.C. Mol. Cell Biol. 1998; 18: 7499-7509Google Scholar, 3Tirasophon W. Welihinda A.A. Kaufman R.J. Genes Dev. 1998; 12: 1812-1824Google Scholar, 4Wang X.Z. Harding H.P. Zhang Y. Jolicoeur E.M. Kuroda M. Ron D. EMBO J. 1998; 17: 5708-5717Google Scholar, 5Haze K. Yoshida H. Yanagi H. Yura T. Mori K. Mol. Biol. Cell. 1999; 10: 3787-3799Google Scholar). They are kept in an inactive monomeric state under nonstressed conditions by binding to the ER chaperone BiP (GRP78). During ER stress, malfolded proteins accumulate in the ER, and BiP is dissociated from the luminal domains of these sensor proteins, leading to their activation (6Bertolotti A. Zhang Y. Hendershot L.M. Harding H.P. Ron D. Nat. Cell Biol. 2000; 2: 326-332Google Scholar, 7Shen J. Chen X. Hendershot L. Prywes R. Dev. Cell. 2002; 3: 99-111Google Scholar). For ATF6, dissociation from BiP allows its translocation to the Golgi, where its cytosolic domain encoding a transcription factor, is cleaved by the site 1 (S1P) and site 2 (S2P) proteases (8Ye J. Rawson R.B. Komuro R. Chen X. Dave U.P. Prywes R. Brown M.S. Goldstein J.L. Mol. Cell. 2000; 6: 1355-1364Google Scholar). The liberated ATF6 then migrates to the nucleus, where it directly binds to and activates the consensus ER stress response element (ERSE) found in the promoters of various UPR targets including transcription factors XBP-1 and CHOP, and ER chaperones like BiP and GRP94 (9Yoshida H. Haze K. Yanagi H. Yura T. Mori K. J. Biol. Chem. 1998; 273: 33741-33749Google Scholar, 10Yoshida H. Okada T. Haze K. Yanagi H. Yura T. Negishi M. Mori K. Mol. Cell Biol. 2000; 20: 6755-6767Google Scholar). For Ire1p, dissociation from BiP leads to its dimerization and activation of the cytosolic kinase domain, which in turns stimulates an endoribonuclease activity located at its C terminus. Activated Ire1 recognizes and cleaves two specific stem loop sequences in the XBP-1 mRNA, which are then religated, resulting in a larger XBP-1 protein (spXBP-1) due to a resulting frameshift (11Calfon M. Zeng H. Urano F. Till J.H. Hubbard S.R. Harding H.P. Clark S.G. Ron D. Nature. 2002; 415: 92-96Google Scholar, 12Shen X. Ellis R.E. Lee K. Liu C.Y. Yang K. Solomon A. Yoshida H. Morimoto R. Kurnit D.M. Mori K. Kaufman R.J. Cell. 2001; 107: 893-903Google Scholar, 13Yoshida H. Matsui T. Yamamoto A. Okada T. Mori K. Cell. 2001; 107: 881-891Google Scholar). Spliced XBP-1 encodes a transcription factor that binds and transactivates the same ERSE site in vitro as ATF6 (13Yoshida H. Matsui T. Yamamoto A. Okada T. Mori K. Cell. 2001; 107: 881-891Google Scholar). However, recent data suggest that not all ATF6 and XBP-1 targets are shared (14Yoshida H. Matsui T. Hosokawa N. Kaufman R.J. Nagata K. Mori K. Dev. Cell. 2003; 4: 265-271Google Scholar, 15Lee A.H. Iwakoshi N.N. Glimcher L.H. Mol. Cell Biol. 2003; 23: 7448-7459Google Scholar). In the case of PERK, BiP dissociation during UPR activation leads to oligomerization and activation of its cytosolic kinase domain. Activated PERK phosphorylates eIF-2α, leading to an immediate, yet transient protein synthesis inhibition (16Harding H.P. Novoa I.I. Zhang Y. Zeng H. Wek R. Schapira M. Ron D. Mol. Cell. 2000; 6: 1099-1108Google Scholar). Unlike ATF6 and Ire1 activation, which are specific to ER stress, the downstream consequences of PERK activation are shared with other cellular stress responses including amino acid deprivation, infection with double-stranded RNA viruses, heme deficiency and oxidative stress (16Harding H.P. Novoa I.I. Zhang Y. Zeng H. Wek R. Schapira M. Ron D. Mol. Cell. 2000; 6: 1099-1108Google Scholar, 17Williams B.R. Oncogene. 1999; 18: 6112-6120Google Scholar, 18de Haro C. Mendez R. Santoyo J. Faseb J. 1996; 10: 1378-1387Google Scholar, 19Brostrom C.O. Brostrom M.A. Prog Nucleic Acids Res. Mol. Biol. 1998; 58: 79-125Google Scholar, 20Kedersha N. Chen S. Gilks N. Li W. Miller I.J. Stahl J. Anderson P. Mol. Biol. Cell. 2002; 13: 195-210Google Scholar, 21Lu L. Han A.P. Chen J.J. Mol. Cell Biol. 2001; 21: 7971-7980Google Scholar). These cellular stresses all activate specific eIF-2α kinases that also cause a transient protein synthesis inhibition. Paradoxically, any condition that causes eIF-2α phosphorylation and the resulting protein synthesis inhibition, will induce ATF4 at the translational level (16Harding H.P. Novoa I.I. Zhang Y. Zeng H. Wek R. Schapira M. Ron D. Mol. Cell. 2000; 6: 1099-1108Google Scholar), leading to activation of its downstream targets (22Ma Y. Brewer J.W. Diehl J.A. Hendershot L.M. J. Mol. Biol. 2002; 318: 1351-1365Google Scholar, 23Ma Y. Hendershot L.M. J. Biol. Chem. 2003; 278: 34864-34873Google Scholar). Therefore, the mammalian ER stress response can be divided into two main branches: one that is downstream of both ATF6 and Ire1p/XBP-1 and is specific to ER stress, and another that is downstream of PERK and is shared by other cellular stresses that activate eIF-2α kinases (24Okada T. Yoshida H. Akazawa R. Negishi M. Mori K. Biochem. J. 2002; 366: 585-594Google Scholar). The C/EBP homologous transcription factor CHOP (GADD153) is the only UPR target that has been shown to be dually regulated by both the ER stress-specific and the shared branches of the UPR (10Yoshida H. Okada T. Haze K. Yanagi H. Yura T. Negishi M. Mori K. Mol. Cell Biol. 2000; 20: 6755-6767Google Scholar, 22Ma Y. Brewer J.W. Diehl J.A. Hendershot L.M. J. Mol. Biol. 2002; 318: 1351-1365Google Scholar, 25Ubeda M. Habener J.F. Nucleic Acids Res. 2000; 28: 4987-4997Google Scholar). The CHOP promoter contains both an ERSE and a C/EBP-ATF composite site, through which these two branches act. Deletion of either element from a CHOP promoter construct leads to a decrease in its transcriptional activity (22Ma Y. Brewer J.W. Diehl J.A. Hendershot L.M. J. Mol. Biol. 2002; 318: 1351-1365Google Scholar). The ATF6 and XBP-1 transcription factors can bind to ERSEs in vitro, as demonstrated by gel shift assays (13Yoshida H. Matsui T. Yamamoto A. Okada T. Mori K. Cell. 2001; 107: 881-891Google Scholar), and ATF6 overexpression leads to CHOP induction in cells (10Yoshida H. Okada T. Haze K. Yanagi H. Yura T. Negishi M. Mori K. Mol. Cell Biol. 2000; 20: 6755-6767Google Scholar). The ATF4 transcription factor binds to the C/EBP-ATF composite site in vitro in an ER stress-dependent manner (22Ma Y. Brewer J.W. Diehl J.A. Hendershot L.M. J. Mol. Biol. 2002; 318: 1351-1365Google Scholar). However, the relative contributions of these two branches in regulating CHOP during different cellular stresses have not been determined. In PERK-null and eIF-2αS51A knock-in cells, where eIF-2α phosphorylation is blocked during ER stress, the induction of CHOP and XBP-1 is completely lost and the up-regulation of BiP is reduced (11Calfon M. Zeng H. Urano F. Till J.H. Hubbard S.R. Harding H.P. Clark S.G. Ron D. Nature. 2002; 415: 92-96Google Scholar, 16Harding H.P. Novoa I.I. Zhang Y. Zeng H. Wek R. Schapira M. Ron D. Mol. Cell. 2000; 6: 1099-1108Google Scholar, 26Scheuner D. Song B. McEwen E. Liu C. Laybutt R. Gillespie P. Saunders T. Bonner-Weir S. Kaufman R.J. Mol. Cell. 2001; 7: 1165-1176Google Scholar). These data support the existence of a cross-talk between the two pathways, where a PERK-dependent target is facilitating the activation of the ATF6 and Ire1/XBP-1-dependent branches of the UPR. Even though the molecular pathway for this cross-talk has not been established, ATF4 was implicated, as it is the only known direct target of eIF-2α phosphorylation during ER stress. Here, we identified Herp, an ER localized protein with an N-terminal ubiquitin-like domain (27Kokame K. Agarwala K.L. Kato H. Miyata T. J. Biol. Chem. 2000; 275: 32846-32853Google Scholar, 28van Laar T. Schouten T. Hoogervorst E. van Eck M. van der Eb A.J. Terleth C. FEBS Lett. 2000; 469: 123-131Google Scholar), to be another UPR target that is dually regulated by both the shared and the ER stress specific branches of the UPR pathway. Using ATF4-null MEFs, we analyzed the relative contributions of the different pathways in controlling CHOP and Herp transcription during ER and other cellular stresses. Our data reveal that although the ATF4-dependent pathway is not required for the induction of these genes during UPR activation, it is essential in response to cellular stresses that activate only the shared branch of the UPR. Furthermore, our data excluded ATF4 as the molecule that is responsible for the cross-talk between the shared and ER stress-specific branches of the UPR. In toto, our results enable us to separate UPR targets into three sets (Fig. 7), those that are specific to the UPR and evidently downstream of ATF6 and Ire1/XBP-1, those that are downstream of PERK and are shared by other cellular stresses, and those that are dually regulated by both pathways. The latter group is likely to be induced by all cellular stresses involving activation of an eIF-2α kinase with maximal levels of induction occuring during UPR activation. DNA Constructs and Chloramphenicol Acetyl Transferase (CAT) Assay—-894 to +35 bp of the mouse Herp promoter was cloned immediately upstream of the CAT reporter gene via a PCR-based method (22Ma Y. Brewer J.W. Diehl J.A. Hendershot L.M. J. Mol. Biol. 2002; 318: 1351-1365Google Scholar) using primer pair 5′-CTCTCCTATCACCTCTCTGAC and 5′-CTGCGTCGCTGGCGGCTC. The C/EBP-ATF composite site (-183 to -176bp) was deleted in the mutant promoter construct. 1 μg of the wild-type or mutant Herp promoter construct was transfected into COS-1 cells, and cell lysates were analyzed for CAT activity as detailed previously (22Ma Y. Brewer J.W. Diehl J.A. Hendershot L.M. J. Mol. Biol. 2002; 318: 1351-1365Google Scholar). Each experimental group was analyzed in triplicate, normalized against protein concentration, and the experiment was repeated twice. The error bars shown in Fig. 2A represent the standard deviations for each set of triplicates. A wild-type eIF-2α cDNA clone was kindly provided by Dr. John W. B. Hershey (University of California, Davis). A point mutant mimicking the phosphorylated form of eIF-2α (S51D), therefore constitutively blocking protein translation, was made with the QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) and sequenced before subcloning both constructs into the pCMV5 vector. Cell Culture, Transfection, and Stress Induction—Primary wild-type and ATF4-/- MEFs were harvested and propagated in tissue culture as described (23Ma Y. Hendershot L.M. J. Biol. Chem. 2003; 278: 34864-34873Google Scholar). Passage 5 through 7 MEFs were plated and left untreated (control), treated with thapsigargin (Tg, 1 μm), or cultured in media that lacked leucine (amino acid deprivation) for the indicated periods of time. Components of the leucine-free media are based on Dulbecco's modified Eagle's medium media (cat. no. 11960), except that leucine is omitted. 293T cells were grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 2 mm glutamine, and 5 ml/liter Fungizone (Mediatech, Herdon, VA). 293T cells were transfected with the indicated vectors using the calcium precipitation method, and cell lysates were harvested 24 h later as described. Isolation of Cytosolic mRNA, Northern Blotting, and Semi-quantitative RT-PCR—Cytoplasmic mRNA was harvested from wild-type and ATF4-/- MEFs, and 293T cells using the Qiagen RNeasy kit (Valencia, CA). 20 μg of cytoplasmic mRNA from each experimental group was electrophoresed, analyzed by Northern blotting as described (29Brewer J.W. Cleveland J.L. Hendershot L.M. EMBO J. 1997; 16: 7207-7216Google Scholar). The human GAPDH probe was purchased from Clontech (Palo Alto, CA). The hamster BiP and mouse ATF4 probes were prepared as described (23Ma Y. Hendershot L.M. J. Biol. Chem. 2003; 278: 34864-34873Google Scholar). The mouse CHOP probe was amplified with the primer pair 5′-GGGAGCTGGAAGCCTGGTAT and 5′-TGCAGGGTCACATGCTTGGC, and the human Herp probe was produced using the primer pair 5′-CCAAGGCCTGGGGCCTGG and 5′-CTGTCGAGTCCACGCCAGG. Total XBP-1 mRNA was detected with a mouse probe amplified with the primer pair 5′-CGGCCTTGTGGTTGAGAACC and 5′-ACGAAAGAGACAGGCCTATGC. Where indicated, the 32P signals were quantified with Molecular Dynamics Phosphorimager (Amersham Biosciences). To quantitate specific mRNAs by RT-PCR, 1 μg of RNA was used to first generate 50 μl of cDNA using a poly(dT) primer (1 mm). Semiquantitative PCR was performed with the Failsafe PCR system (Epicenter, Madison, WI) using 2 μl of cDNA and [α-32P]dCTP (23Ma Y. Hendershot L.M. J. Biol. Chem. 2003; 278: 34864-34873Google Scholar). The labeled PCR products were separated on a 4% TBE-acrylamide gel, dried, and exposed to film. Quantitative data was obtained using a Molecular Dynamics phosphorimager and was normalized to the level of the γ-actin transcript measured from the same sample. The linear range was monitored by using serial dilutions (1:5 and 1:25) of the sample that showed the highest radioactive signal in each experimental group. The primer pair for mouse γ-actin was: 5′-CCGACGGGCAGGTGATCAC and 5′-GAGCAGTTAACTTGAATACAAGG, and for the mouse spXBP-1, 5′-GCTGAGTCCGCAGCAGGTG and 5′-ACGAAAGAGACAGGCCTATGC were used. Western Blot—For direct Western blotting, cells were lysed in Nonidet P-40 lysing buffer and proteins were electrophoresed under reducing conditions, transferred to a nitrocellulose membrane, and probed with the indicated primary antibodies: rabbit anti-BiP and anti-CHOP (22Ma Y. Brewer J.W. Diehl J.A. Hendershot L.M. J. Mol. Biol. 2002; 318: 1351-1365Google Scholar), rabbit anti-ATF4 and goat anti-Hsc70 (Santa Cruz Biotechnology), and monoclonal mouse anti-pan eIF-2α (BIOSOURCE, Camarillo, CA). Nuclear Extracts and Electrophoretic Mobility Shift Assays—Wild type or ATF4-null MEFs were treated with Tg (1 μm) for the indicated times, harvested, and washed with phosphate-buffered saline twice before nuclear extracts were prepared (22Ma Y. Brewer J.W. Diehl J.A. Hendershot L.M. J. Mol. Biol. 2002; 318: 1351-1365Google Scholar). The EMSA reaction was performed with the appropriate [γ-32P]ATP-labeled probe (10,000 cpm) as introduced before (22Ma Y. Brewer J.W. Diehl J.A. Hendershot L.M. J. Mol. Biol. 2002; 318: 1351-1365Google Scholar). When indicated, 2 μg of a supershift antibody (polyclonal anti-ATF4 antisera, Santa Cruz Biotechnology) was added to the reaction. The complimentary strands of the oligonucleotide probes were annealed and end-labeled with [γ-32P]ATP. The 29-bp CHOP C/EBP-ATF composite site probes (both wild type and mutant) were synthesized as before (22Ma Y. Brewer J.W. Diehl J.A. Hendershot L.M. J. Mol. Biol. 2002; 318: 1351-1365Google Scholar). Similarly, a 29-bp wild-type sequence containing -191 bp to -167 bp of the human Herp promoter (5′-GATACGCGGCGGGTTGCATCAGCCCGTGA) was synthesized and labeled. For the mutant Herp oligonucleotides, the underlined sequence of the wild-type probe containing the ATF site was altered to ccgtCtctAc. Identification of a Conserved C/EBP-ATF Composite Site in the Herp Promoter—To determine if the regulation of CHOP was unique among UPR targets, we searched the promoters of other genes up-regulated during ER stress for elements that might suggest they were also dually regulated. We identified the presence of a potential C/EBP-ATF composite site in the promoter of an ER-localized protein, Herp (Fig. 1). This site is located just upstream of the ERSE I and II sites that were previously characterized in the Herp promoter (28van Laar T. Schouten T. Hoogervorst E. van Eck M. van der Eb A.J. Terleth C. FEBS Lett. 2000; 469: 123-131Google Scholar, 30Kokame K. Kato H. Miyata T. J. Biol. Chem. 2001; 276: 9199-9205Google Scholar) and is completely conserved between the human and mouse genes. To determine if the C/EBP-ATF composite site contributed to the transcriptional up-regulation of Herp during ER stress, we isolated a 930-bp fragment of the murine Herp promoter containing ERSE I, ERSE II, and the potential C/EBP-ATF composite sites and tethered it to a CAT reporter. The wild-type promoter showed low basal activity and was induced ∼12-fold when the cells were treated with UPR inducing agents, demonstrating that this DNA fragment contained the necessary elements for responding to ER stress. Mutation of the composite site alone in the context of the murine Herp promoter construct did not dramatically alter the basal level of transcription but did significantly decrease its transcriptional activity after ER stress (Fig. 2A). To determine if ATF4 could bind directly to the Herp C/EBP-ATF composite site upon UPR activation, we synthesized wild-type and mutant oligonucleotides encompassing the composite site and performed electrophoretic mobility shift assays (EMSA) with nuclear extracts harvested from both wild-type and ATF4-null primary MEFs that were left untreated or incubated with thapsigargin to induce ER stress (Fig. 2B). Oligonucleotides encompassing the CHOP composite site, which are the same length as those synthesized for Herp, were used as a positive control. As expected, ATF4 bound to the CHOP probe in an ER stress-dependent manner when nuclear extracts from wild-type MEFs were used (Fig. 2B, lane 3) and could be supershifted with an antiserum to ATF4 (lane 4). When the Herp probe was similarly analyzed, we observed a complex (a) that co-migrated with the one that bound to the CHOP probe. Although trace amounts of this complex formed in the absence of ER stress (lane 5), the signal dramatically increased after ER stress (lane 7), and in both cases it was supershifted with the ATF4 antibody (lanes 6 and 8). A fainter, faster migrating complex (*), which also contained ATF4 binding activity, was observed with the Herp (lanes 7 and 8), but not the CHOP probe. This difference could either be due to the fainter signal obtained with the CHOP probe, or it is possible that it is specific to Herp. When nuclear extracts harvested from ATF4-null MEFs were used in the gel shift reactions (lanes 9–16), no binding activity was detected with either probe that could be supershifted with the ATF4 antibody, further demonstrating that the binding activity detected in the wild-type nuclear extracts was dependent on the presence of ATF4. Hence, we conclude that Herp, like CHOP, represents another UPR target that is dually regulated by both the shared cellular stress response and the UPR-specific pathways during ER stress. eIF-2α Phosphorylation Alone Was Sufficient to Induce Herp and CHOP, but Not BiP—To further determine if Herp was a direct target of the PERK/eIF-2α/ATF4-dependent pathway and could be induced independent of ATF6 and XBP-1 activation, we transfected cells with GFP, wild-type eIF-2α, or the eIF-2αS51D mutant, which conformationally and functionally imitates the phosphorylated form of eIF-2α and as a result inhibits protein synthesis (31Choi S.Y. Scherer B.J. Schnier J. Davies M.V. Kaufman R.J. Hershey J.W. J. Biol. Chem. 1992; 267: 286-293Google Scholar). Cells expressing GFP alone served as a control for basal and stress-induced expression of UPR targets (Fig. 3). The transfection itself did not induce ER stress, and the cells were still capable of inducing UPR targets like ATF4 and CHOP when treated with thapsigargin (Fig. 3, A and B). When cells transfected with the eIF-2α constructs were similarly examined, we found that expression of the constitutively active eIF-2α S51D mutant led to the robust induction of ATF4 and CHOP proteins, whereas expression of the wild-type eIF-2α construct caused only a very slight induction of these targets. To more closely examine the induction of UPR targets, mRNA was harvested from these cells and Northern blot analyses were performed (Fig. 3B). In keeping with the Western blot data, expression of the eIF-2α S51D mutant, but not the wild-type eIF-2α construct, resulted in Herp and CHOP induction, although to a level significantly lower than that obtained with thapsigargin treatment. Transcription of BiP, another UPR target, was not significantly altered by either wild-type or mutant eIF-2α expression, demonstrating that the ATF6 and Ire1/XBP-1 arms of the UPR were not affected. ATF4 Was Not Essential for Herp and CHOP Induction during ER Stress, but Was Required for Their Optimal Activation—We wished to measure the relative contribution of the ER-specific and the shared UPR pathways in the induction of both Herp and CHOP. First, we compared the mRNA levels of these two genes in the wild-type and ATF4-null cells during UPR activation, where both the shared (PERK-dependent) and the ER stress-specific (ATF6 and XBP-1-dependent) arms are activated. The thapsigargin-mediated induction of both CHOP and Herp transcripts in ATF4-null cells was reduced to ∼35% of that observed in wild-type cells, but was still very much apparent (Fig. 4A). The accumulation of CHOP protein in response to ER stress was also decreased in the ATF4-null cells, but could still be easily detected (Fig. 4B), demonstrating that although ATF4 clearly contributes to the transcription of these two genes during UPR activation, it is not completely essential. This result is different from a previous Western blot study where CHOP protein induction during ER stress was completely abolished in their ATF4-null MEFs (32Harding H.P. Zhang Y. Zeng H. Novoa I. Lu P.D. Calfon M. Sadri N. Yun C. Popko B. Paules R. Stojdl D.F. Bell J.C. Hettmann T. Leiden J.M. Ron D. Mol. Cell. 2003; 11: 619-633Google Scholar). The phenotypes of the two ATF4-null mice are very similar with both showing lens development defect and with the majority of homozygotes dying between E14.5 and birth (our ATF4-/- mice birth rate: 6.5% versus Dr. J. M. Leiden, 7.5%) (23Ma Y. Hendershot L.M. J. Biol. Chem. 2003; 278: 34864-34873Google Scholar, 33Tanaka T. Tsujimura T. Takeda K. Sugihara A. Maekawa A. Terada N. Yoshida N. Akira S. Genes Cells. 1998; 3: 801-810Google Scholar, 34Hettmann T. Barton K. Leiden J.M. Dev. Biol. 2000; 222: 110-123Google Scholar). Combined with the facts that in our ATF4-/- MEFs, 58% of the coding region of ATF4 including the entire basic DNA binding domain and the leucine-zipper dimerization domain was substituted with a neomycin resistance gene, no ATF4 transcript was detected by Northern blotting (Fig. 4A), and no ATF4 protein was detected by either Western blotting (23Ma Y. Hendershot L.M. J. Biol. Chem. 2003; 278: 34864-34873Google Scholar) or gel shift assays (Fig. 2B), we believe that the difference between the two experiments could be a result of differences in the sensitivities of the two CHOP antisera. We next determined if the ATF4 effects we observed on the transcription of these two genes was exerted entirely through the ATF composite site, or if ATF4 could be the PERK-dependent target that facilitates the activation of the Ire1/XBP-1 and ATF6-dependent branches of the UPR as previously suggested (11Calfon M. Zeng H. Urano F. Till J.H. Hubbard S.R. Harding H.P. Clark S.G. Ron D. Nature. 2002; 415: 92-96Google Scholar, 16Harding H.P. Novoa I.I. Zhang Y. Zeng H. Wek R. Schapira M. Ron D. Mol. Cell. 2000; 6: 1099-1108Google Scholar, 26Scheuner D. Song B. McEwen E. Liu C. Laybutt R. Gillespie P. Saunders T. Bonner-Weir S. Kaufman R.J. Mol. Cell. 2001; 7: 1165-1176Google Scholar). We attempted to transfect both wild-type and ATF4-null MEFs with a CHOP promoter construct that contains the ERSEs, but not the ATF composite site, tethered to a reporter gene (22Ma Y. Brewer J.W. Diehl J.A. Hendershot L.M. J. Mol. Biol. 2002; 318: 1351-1365Google Scholar). Unfortunately, we were unable to achieve a high enough transfection efficiency to get a measurable signal in either of the cells. As an alternative approach, we treated wild-type and ATF4-null MEFs with thapsigargin to induce ER stress and compared the induction of BiP and total XBP-1 transcripts, which are downstream of ATF6 (9Yoshida H. Haze K. Yanagi H. Yura T. Mori K. J. Biol. Chem. 1998; 273: 33741-33749Google Scholar, 10Yoshida H. Okada T. Haze K. Yanagi H. Yura T. Negishi M. Mori K. Mol. Cell Biol. 2000; 20: 6755-6767Google Scholar). We found no evidence to suggest that the induction of BiP was reduced in the ATF4-null cells, but instead found that the total XBP-1 transcripts were actually slightly increased (Fig. 5A). We then designed PCR oligonucleotides that specifically amplified only the spliced form of the XBP-1 transcript, and semi-quantitative RT-PCR reactions were performed to measure XBP-1 splicing. We found that XBP-1 splicing, which is mediated by the activated Ire-1 endoribonuclease domain, was also not inhibited in the absence of ATF4 (Fig. 5B). A comparison of quantitative data from three different experiments showed that again there was a slight increase in the activity of this arm of the UPR in the ATF4-null cells (Fig. 5C). Finally, we accessed the induction of ERdj4 (35Shen Y. Meunier L. Hendershot L.M. J. Biol. Chem. 2002; 277: 15947-15956Google Scholar), a recently identified DnaJ homologue that is regulated by XBP-1, not ATF6 (15Lee A.H. Iwakoshi N.N. Glimcher L.H. Mol. Cell Biol. 2003; 23: 7448-7459Google Scholar). In keeping with the slightly higher levels of total XBP-1 transcripts and the increased splicing of XBP-1 in the ATF4-null cells, our data showed that the induction of ERdj4 was also somewhat higher in the absence of ATF4 (Fig. 5A). The kinetics of ERdj4 induction are somewhat delayed compared with XBP-1 induction and splicing, which is consistent with it being an XBP-1 target (15Lee A.H. Iwakoshi N.N. Glimcher L.H. Mol. Cell Biol. 2003; 23: 7448-7459Google Scholar). Therefore, during ER stress, the attenuated CHOP and Herp induction in ATF4-null MEFs must be due to a loss of activation at the composite site and not to changes in the ATF6 and Ire1/XBP-1 pathways that converge at the ERSE site. Our data reveal that these pathways are completely intact, and even a bit more vigorous, in the ATF4-null animals. ATF4 Was Required for the Activation of CHOP and Herp by Cytosolic Stresses That Activate Other eIF-2α Kinases like GCN2—We next determined the role of ATF4 in the induction of Herp and CHOP by cellular stresses that activate other eIF-2α kinases. We incubated both wild-type and ATF-null MEFs with medium lacking leucine to induce amino acid deprivation stress. This treatment does not activate the ATF6 and