The FASEB Journal express article 10.1096/fj.02-1184fje. Published online June 3, 2003.
The function of GADD34 is a recovery from a shutoff of protein synthesis induced by ER stress—elucidation by GADD34-deficient mice Eiji Kojima,*,† Akihide Takeuchi,* Masataka Haneda,* Fumiko Yagi,*,† Tadao Hasegawa,* Ken-ichi Yamaki,† Kiyoshi Takeda,‡ Shizuo Akira,‡ Kaoru Shimokata,† and Ken-ichi Isobe*,¶ *Department of Basic Gerontology, National Institute for Longevity Sciences, 36-3 Gengo, Morioka-cho, Obu, Aichi 474-8522, Japan; †Department of Internal Medicine, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, Aichi 466-8520, Japan; and ‡ Department of Host Defense, Research Institute for Microbial Diseases, Osaka University ¶
Corresponding author: Ken-ichi Isobe, Department of Basic Gerontology, National Institute for Longevity Sciences, 36-3, Gengo, Morioka-cho, Obu, Aichi 474-8522, Japan. E-mail:
[email protected] ABSTRACT GADD34 is a protein that is induced by stresses such as DNA damage. The function of mammalian GADD34 has been proposed by in vitro transfection, but its function in vivo has not yet been elucidated. Here we generated and analyzed GADD34 knockout mice. Despite their embryonic stage- and tissue-specific expressions, GADD34 knockout mice showed no abnormalities at fetal development and in early adult life. However, in GADD34–/– mouse embryonic fibroblasts (MEFs), recovery from a shutoff of protein synthesis was delayed when MEFs were exposed to endoplasmic reticulum (ER) stress. The phosphorylation of eukaryotic translation initiation factor 2 α (eIF2α) at Ser51 induced by thapsigargin or DTT was prolonged in GADD34–/– MEF, although following treatment with tunicamycin, the eIF2α phosphorylation level did not change in either GADD34+/+ or GADD34–/– cells. ER stress stimuli induced expressions of Bip (binding Ig protein) and CHOP (C/EBP homologous protein) in MEF of wild-type mice. These expressions were strongly reduced in GADD34–/– MEF, which suggests that GADD34 up-regulates Bip and CHOP. These results indicate that GADD34 works as a sensor of ER stress stimuli and recovers cells from shutoff of protein synthesis. Key words: unfolded protein response • translational attenuation • eukaryotic initiation factor
G
ADD34 is a member of the protein family whose expression is up-regulated by growth arrest and DNA damage (1). GADD34, like GADD45 and GADD153, was originally discovered as an UV-inducible transcript in Chinese hamster ovary cells (2). From extensive studies, GADD45 is reported to be regulated by p53 and is a key player in the maintenance of genomic stability (3,4). GADD153, also referred to as CHOP (C/EBP homologous protein), is one of the C/EBP transcription factors and has been shown to induce apoptosis (5–7). Although several groups have investigated the function of GADD34 by in vitro experiments, its true function remains unclear, especially in vivo. GADD34 is also induced by amino acid deprivation and several endoplasmic reticulum (ER) stresses (8, 9). GADD34 harbors a highly
conserved domain in its carboxyl-terminus, which is homologous to γ134.5 of HSV1. γ134.5 is a virulence factor that blocks the premature shutoff of protein synthesis in HSV1-infected neuroblastoma cells (10), and this block may interfere with apoptosis. The carboxyl-terminal domain of the γ134.5 protein binds to protein phosphatase 1α (PP1). This complex specifically dephosphorylates eukaryotic translation initiation factor 2 α (eIF2α), which is evolved to preclude a shutoff of protein synthesis (11). Several studies demonstrated that the onset of apoptosis is correlated with GADD34 expression in selected cell lines after ionizing irradiation or alkylating agent of methyl methanesulfate treatment (12,13). Several proteins have been shown to be associated with GADD34. An association with proliferating cell nuclear antigen suggests that GADD34 might inhibit proliferation (14). We have shown that GADD34 interacts with Zfp148 (also known as BFCOL1), which might affect p21/WAF1 transcription (15). HRX leukemic fusion oncogenes, the human homologue of the Drosophila trithorax (trx) gene, binds GADD34 to negatively regulate the apoptotic response (12). The expression of GADD34 in the colorectal cancer cell line SW480 has been reported to enhance IR-induced apoptosis (12). Although GADD34 expression is induced by various stress stimuli (IR, MMS, etc.), it is also induced by other stimuli, including IL-6 and ROS (16, 17). These facts may indicate that GADD34 has some function in development and differentiation. In order to understand the function of GADD34, we first examined its expression in fetal and normal adult tissues, and then we disrupted GADD34 gene. We show that GADD34 expression is stage-specific in fetal development and organ-specific in adult mice. Despite that GADD34-deficient mice showed no obvious abnormalities in either newborns or adult mice, GADD34–/– mouse embryonic fibroblasts (MEFs) show delayed recovery from the shutoff of protein synthesis by ER stresses, suggesting that GADD34 plays important roles in these situations. MATERIALS AND METHODS Materials Thapsigargin (Tg) and methyl methanesulfonate (MMS) were purchased from Sigma-Aldrich (St. Louis, MO). Tunicamycin (Tm) and dithiothreitol (DTT) were from Calbiochem-Novabiochem and WAKO (Japan), respectively. Tg stock solutions were freshly prepared with DMSO, and other agents with sterilized water. Male C57BL/6CR mice, 2 months old, were obtained from Nihon SLC. Northern blot analysis Total RNA was extracted from mouse tissues or MEFs, and about 20 µg of each total RNA preparation was electrophoresed on 1% agarose gels containing 1.1 M formaldehyde. The RNA was transferred to Genescreen Plus membrane (NEN Life Science, Boston, MA). mRNA was detected with a 32P-labeled GADD34 (random primer labeling kit; Takara, Ohtsu, Japan) after hybridization for 18 h at 42 °C in 5 × SSPE (1 × SSPE is composed of 0.18 M NaCl, 10 mM sodium phosphate (pH 7.7), and 1 mM EDTA), 5 × Denhardt's solution (1 × Denhardt's solution is composed of 0.02% bovine serum albumin, 0.02% Ficoll, and 0.02% polyvinylpyrrolidone), 50% formamide, 0.1% SDS, 50 µg/ml heat-denatured salmon testis DNA, and a radioactive probe. Membranes were washed twice for 15 min each at 65 °C in a solution containing 2 × SSC (1 × SSC is composed of 0.15 M NaCl and 15 mM sodium citrate) and 0.1% SDS, then once in 1 × SSC with
0.1% SDS for 30 min at 65 °C and finally twice for 15 min each in 0.1% SSC with 0.1% SDS at room temperature. Signals were detected by the BAS2500 system. The probes used here were as follows: 1678-2077 bp portion of GADD34, 175-681bp portion of CHOP, and 539-872bp portion of Bip. Human 28S rRNA or mouse β-actin probe was used as an internal RNA-loading control. Immuunoblot analysis Cells were scraped off the dishes with lysis buffer [20 mM Hepes (pH 7.5), 1% Triton X-100, 150 mM NaCl, 10% glycerol, and 1 mM EDTA containing 1 mM phenylmethylsulfonyl fluoride (PMSF), leupeptin (1 µg/ml), and pepstatin (1 µg/ml)]. The extracts were centrifuged at 12,000 x g for 15 min at 4 °C, and the supernatants were boiled for 5 min in the sampling buffer [100 mM Tris-HCl (pH 6.8), 4% SDS, 20% glycerol, 150 mM β-mercaptoethanol, 1% bromophenyl blue]. About 20 µg of protein was electrophoresed on a 9 or 12% SDS/PAGE, and then transferred to nitrocellulose membranes. The blots were blocked with PBS-T (1 × PBS with 0.05% Tween20) containing 5% skim milk for 1 h, and incubated with anti-GADD34 (Santa Cruz) rabbit polyclonal immunoglobulin G for 2 h at room temperature, washed three times, and incubated with HRP-conjugated mouse monoclonal IgG. (Santa Cruz). Signals were detected by the ECL system (Amersham Pharmacia Biotech). The following four primary antibodies from Santa Cruz Biotechnology were used: rabbit anti-GADD34, rabbit anti-GADD153 (CHOP), rabbit anti-GRP78 (BiP), goat anti-eIF-2α, and rabbit anti-ATF4 together with rabbit anti-eIF-2α-P (Research Genetics, Huntsville, AL) Generation of GADD34 knockout mice The genomic DNA fragment containing the GADD34 gene was screened from a 129/SvJ mouse genomic library (Stratagene, La Jolla, CA), subcloned into pBluescript (Stratagene), and characterized by restriction enzyme mapping and DNA sequencing. A targeting vector was constructed to replace the 0.2 kb genomic fragment containing a part of the second exon with the neomycin resistance gene from pMC1-neo (Stratagene). A herpes simplex virus thymidine kinase gene driven by the MC1 promoter was used for negative selection. The targeting vector was linearized with SalI and electroporated into E14.1 embryonic stem (ES) cells. ES cell clones that were resistant to both G418 and gancycrovir were screened for homologous recombination by PCR and confirmed by Southern blot analysis. Chimeric mice were generated by microinjection of the targeted ES cell clones into blastocysts obtained from C57BL/6 females to produce heterozygous mice. Heterozygous mice were interbred to generate homozygotes. GADD34-deficient mice and their wild-type littermates from these intercrosses were used for experiments. Histological analysis and in situ hybridization Embryos, 12.5 days old, were fixed with 4% paraformaldehyde, dehydrated, and embedded in paraffin. In situ hybridization was performed as previously described (18) with modifications. Specific DIG-labeled antisense and sense RNA probes (1678–2077 bp portion of GADD34) were prepared by using the RiboProbe In Vitro Transcription Systems (Invitrogen). The RNA probe was visualized with anti-DIG-AP Fab fragment (Roche) and BCIP/NBT solutions.
Preparation of mouse embryonic fibroblasts MEFs were prepared from 14.5-day-old embryos. All cultures were maintained in Dulbecco’s modified essential medium (Sigma) with 10% fetal calf serum (FCS). Cells were plated 1–2 × 10 6 /10 cm plate for subculture and experimentation. Plasmids and transfection Total RNA was isolated from NIH3T3 cells and reverse-transcribed into cDNA. The following primers were used for reverse transcription-PCR (RT-PCR) of the full-length murine GADD34 cDNA: 5’-GGAATTCCAGACACATGGCCCCGAGC-3’ and 5’-CGCGGATCCGCCCCGCCTCCCTCCCAAG-3’. The PCR products were cut with EcoRI/BamHIand inserted into pcDNA3.1/Myc-His A vector (Invitrogen). The cloned construct was sequenced and confirmed to be GADD34. MEFs were transiently transfected by using Superfect Transfection Reagent (QIAGEN), incubated for 24 h posttransfection, and exposed to ER stress. Analysis of protein synthesis Analysis of protein synthesis was performed as previously described (20). ER stress inducer-treated cells were labeled with Redivue [35S] methionine (10 mCi/ml; Amersham) for 30 min in a methionine-free minimal essential medium (Invitrogen). After being washed with phosphate-buffered saline, cell extracts were prepared by lysing the cells with Nonidet P-40 lysis buffer (2% Nonidet P-40, 80 mM NaCl, 100 mM Tris-HCl, 0.1% SDS). The protein concentration was measured by using the Bio-Rad protein DC assay kit. Equal amounts (10 µg) of total protein from cell lysates were resolved by 12% SDS-PAGE. The nascent protein synthesis was detected and analyzed by the BAS2500 system. RESULTS GADD34 expression is highly expressed at the fetal stage and in adult mouse organs. At the fetal stage, GADD34 mRNA expression was detected on embryonic day 8.5 (E8.5) and E9.5. Almost no expression was detected on E10.5 or E11.5. Its expression reappeared and was strongest on E12.5, decreased on E16.5, and reappeared on E18.5 (Fig. 1a). The distribution of GADD34 mRNA expression on E12.5 was ubiquitous, but was especially strong in the brain, spinal cord, tongue, lung, and genital tubercle. (Fig. 1b). In adult mice, GADD34 expression was strong in the spleen and lung, moderate in the thymus and muscle, weak in the brain, and almost undetectable in the other organs examined (liver, small intestine, testis, and ovary) (Fig. 1c). Generation of GADD34 knockout mice To elucidate the role of GADD34 in vivo, we generated GADD34–/– mice. We replaced a portion of GADD34, including a translation initiation codon with the neomycin resistance gene from pMC1-neo (Fig. 2a). To facilitate genotyping, PCR was used to detect the endogeneous and mutant genes (Fig. 2b). Western blot analyses were performed with extracts of liver or MEFs from GADD34+/+, +/–, and –/– mice reveals GADD34 was induced in wild or heterozygous mutant by MMS treatment both in vitro and in vivo. These inductions of GADD34 protein were complete absent in homozygous mutant (Fig. 2c, d). Note that transcript from GADD34 cDNA appeared in
similar portion as 100 kDa protein, which indicated that, the antibody specifically detected GADD34 protein. Matings between GADD34 heterozygous mice yielded the expected frequency of wild-type, nullizygous (GADD34–/–), and heterozygous (GADD34+/–) offspring (n=176; 44, 46, and 86, respectively). These results demonstrate that GADD34 deficiency has no severe effects on normal mouse development. GADD34–/– mice also did not show abnormal phenotypes or signs of disease during at least the first 12 months of life. GADD34 induction in the unfolded protein response (UPR) Previous studies demonstrated that rodent GADD34 was inducible by treatment of cell lines with various DNA-damaging agents, such as alkylating agent (21), and also that it was mediated by some of the agents causing UPR (8, 9). Tunicamycin (Tm) inhibits asparagines (N)-linked glycosylation and dithiothreitol (DTT) disrupts disulfide bond formation. Thapsigargin (Tg) is an inhibitor of the Ca2+ ATPase transporter known to rapidly activate the ER stress response. We investigated the levels of GADD34 expression by these ER stress-inducible agents. Tm induced GADD34 in a dosage-dependent manner, whereas Tg and DTT induced GADD34 at over 0.05 µM and 5 mM, respectively, with levels remaining unchanged (Fig. 3a). All three ER stress-inducible agents elevated the level of GADD34 in a time-dependent manner (Fig. 3b). Tg or DTT-induced eIF-2α phosphorylation levels of GADD34–/– MEFs are higher than those of GADD34+/+ MEFs The COOH terminus of GADD34 is homologous to the corresponding domain of the herpes simplex virus encoded protein γ134.5 (10, 11). Both γ134.5 and GADD34 interact with PP1 and dephosphorylate eIF2α (22, 23). To determine whether the expression of GADD34 affects eIF2α phosphorylation levels, we compared those levels of GADD34+/+ MEFs treated with ER stress-inducing agent to those of GADD34–/– MEFs. In wild-type MEFs, the eIF2α phosphorylation level was temporarily increased just after treatment with Tg or DTT, and promptly decreased, whereas in GADD34-deficient MEFs eIF2α is strongly phosphorylated and sustained a high level of phosphorylation (Fig. 4a, b). As initial up-regulation of phosphorylated eIF2α does not differ between GADD34+/+ and –/– MEFs, it is suggested that phosphorylation of eIF2α is GADD34-independent, while its dephosphorylation is GADD34-dependent. By contrast, in case of treatment with tunicamycin, the eIF2α phosphorylation level did not change in either GADD34+/+ or –/– cells (data not shown). Tg-induced eIF2α phosphorylation levels in GADD34–/– MEFs were reduced by exogenously expressed GADD34 To confirm whether prolonged eIF2α phosphorylation in GADD34–/– MEFs by Tg treatment is due to the loss of GADD34 protein, we did the rescue experiment by transiently expressing GADD34 proteins in GADD34–/– MEFs (Fig. 5). We transfected pcDNA3.1Myc-His-GADD34 vector in GADD34–/– MEFs and treated them with Tg. The expression of pcDNA3.1Myc-His-GADD34 vector lead to reduction of eIF2α phosphorylation levels compared with that of empty pcDNA3.1Myc-His vector. This result demonstrated that GADD34 decreased the levels of eIF2α phosphorylation by Tg treatment.
Tg or DTT treatment inhibits protein synthesis in GADD34–/– MEFs eIF2α is a heterotrimeric protein that is required to bring the initiator methionyl-transfer RNA (Met-tRNA) to the 40S ribosome (24). Phosphorylation of eIF2α reduces its functional level and limits initiation translation on all cellular mRNAs within the cells. To clarify whether or not strongly phosphorylated eIF2α in Tg- or DTT-treated GADD34–/– MEFs has an effect on translation, we examined the new protein synthesis rates in the treated MEFs. In both genotypes of MEFs, protein synthesis was extremely reduced just after Tg treatment. Protein synthesis gradually recovered in GADD34+/+ MEFs, while it remained at lower levels even after 12 h of Tg treatment in GADD34–/– MEFs (Fig. 6a). By treatment with another ER stress agent, DTT, GADD34–/– cells also kept protein synthesis at a low level until 2 h after treatment (Fig. 6b), whereas tunicamycin did not change the translation rates (data not shown). These results indicate that highly phosphorylated eIF2α in GADD34–/– MEFs leads to a reduction of newly synthesized protein. Tg-induced expression of BiP/GRP78 (glucose regulated protein of molecular weight 78 kDa) and CHOP/GADD153 are delayed in GADD34–/– MEFs Bip/GRP78 and CHOP /GADD153 have been shown to be downstream target proteins in ER stress responses (8). Bip, which is a classical marker for UPR activation (25), plays a role as chaperone in the UPR. CHOP has been reported as an ER stress-associated apoptosis factor (5–7). We analyzed Bip and CHOP expressions in GADD34-deficient MEF under ER stress. Although both mRNAs and proteins of Bip and CHOP are strongly induced by Tg treatment in wild-type MEFs, they are weakly induced by Tg treatment in GADD34-deficient MEFs (Fig. 7a, b). Phosphorylation of eIF2α is required for ATF4 (activating transcription factor 4) protein expression during ER stress (26). Transcription of Bip and CHOP are both activated by ATF4 through binding their AARE (amino acid-response element) (27). Then we also analyzed the level of ATF4 protein between Tg-treated GADD34+/+ and –/– MEFs. ATF4 protein levels in wild-type MEF is higher than that of GADD34-deficient MEF (Fig. 7a). These result indicates that GADD34 exists upstream of ATF4, which activates BiP and CHOP. DISCUSSION We have shown in this paper that GADD34 is expressed in normal tissues in an unstimulated state and that its expression in the fetus is unexpected. At the fetal stage, GADD34 expression is especially strong on E12.5. Almost no expression was detected on E10.5 and E11.5, although GADD34 was expressed on E8.5 and E9.5. It is of interest to know why GADD34 expression is different in the fetal stages. The expression of GADD34 is up-regulated by several stimulations including ROS (17) and also by IL-6 (16). The differences in stage-specific expression in the fetus may depend on the stage-specific expression of ligands (growth factors), which induce the expression of GADD34 or ROS, whose production may differ in fetal development. Fetal stage-specific expression of GADD34 is similar to that of Zfp148 (BFCOL1, ZBP-89) (Takeuchi; manuscript in preparation). GADD34 and BFCOL1 interact by yeast two-hybrid methods (19). Despite the stage-specific expression of GADD34, mice deficient in it have no abnormalities under normal breeding conditions. On the contrary, Zfp148 deficiency shows a severe phenotype (28). Other proteins, which have functional similarity to GADD34, may compensate for the GADD34 functions.
Although, GADD34–/– mice show a normal phenotype, ER stress induces dramatic differences in MEF between a wild-type and a GADD34 deficiency. We show that GADD34 reverts the phosphorylation of eIF2α induced by Tg or DTT. These treatments, which induce an ER stress response, shutoff the protein synthesis of both GADD34-deficient and wild-type MEF. However, in wild-type MEF an early recovery from the shutoff is observed, which correlates with the GADD34 expression. In the present study Tm, despite the agent that disrupts protein folding in the ER, induce neither the phosphorylation of eIF2α nor shutoff of the protein synthesis. These results may be caused by the difference of its mechanism in the UPR. It has been shown that cells expressing GADD34 by in vitro transfection experiments diminish the phosphorylation of eIF2α levels in response to ER stress (8). GADD34 forms a complex with the catalytic subunit of PP1 that specifically promotes the dephosphorylation of eIF2α (23). These results correlate with the function of γ134.5 of HSV, which has been found to combine with PP1 (22) and dephosphorylates eIF2α, thereby precluding the shutoff of protein synthesis (29). The thermostable protein inhibitor 1 (I-1), a protein kinase A (PKA)-activated inhibitor of PP1, also binds to GADD34, and the PP1/GADD34/I-1 complex regulates the dephosphorylation of eIF2α (23). In mammalian cells, there are at least four different eIF2α kinases, including GCN2 (30), PKR (31, 32), PERK (33, 34), and HRI (35), which are activated by amino acid starvation, viral infection, endoplasmic reticulum stress, and heme depletion, respectively. It has been shown that GADD34 induction by tunicamycin is absent in PERK–/– cells, and GADD34 induction by amino acid is absent in GCN2–/– cells (8). These reports indicate that GADD34 may be located downstream from signaling of PERK or GCN2 protein. We have shown in this paper that CHOP and BiP are strongly induced by ER stresses in wild-type MEF, although this induction is weak in GADD34-deficient MEF (Fig. 7). Novoa et al. have shown that CHOP and BiP activation are impaired in a tunicamycin-treated COOH fragment of hamster GADD34 (292-590 amino acids)-transduced cells (8). Such contradictory results may derive from the fact that, although our GADD34–/– MEF is deficient in complete GADD34, Novoa et al. use a C terminal fragment of GADD34. It has been reported that transcriptional activation of CHOP and BiP upon activation of the UPR requires eIF2α phosphorylation (26). We have shown in this paper that both transcriptional and translational expression of CHOP and BiP are strongly induced by ER stresses in wild-type MEF, although this induction is weak in GADD34-deficient MEF, despite sustaining a high level of phosphorylated eIF2α. We also have shown that ATF4, which regulates CHOP and Bip transcription, is up-regulated by ER stress in wild-type MEF. The up-regulation of ATF4 in GADD34-deficient MEF is lower than that of wild-type. These results suggest that there exists positive pathway from GADD34 to CHOP or BiP expression through ATF4 in response to ER stress. Although translational attenuation caused by ER stress has been discussed in great detail (24), a recovery from shutoff of protein synthesis has not studied a lot. What is the biological significance of recovery from translational attenuation in response to ER stress? GADD34, which functions as a recovery from a shutoff of protein synthesis, may be important for the maintenance of the homeostasis of cells in the UPR. Recent works gave us possible roles of the recovery from a shutoff of protein synthesis caused by GADD34 in vivo. It has been reported recently that GADD34 is expressed in the peri-infarct zone after focal cerebral ischemia in rats (36). It has been also reported that mammalian eIF2α kinase-dependent autophagy is antagonized by the herpes simplex virus (HSV)-encoded neurovirulence gene product, ICP34.5 (37). Further analysis of the
GADD34 engaged-molecular pathway of the recovery from a shutoff of protein synthesis will open the new field in the pathophysiology of the mammalian system. ACKNOWLEDGMENTS This study was supported by the Fund for Comprehensive Research on Aging and Health, and in part by a grant from the Ministry of Education, Science, Sports and Culture of Japan. REFERENCES 1. Zhan Q., Lord, K. A., Alamo, I., Jr., Hollander, M. C., Carrier, F., Ron, D., Kohn, K. W., Hoffmann, B., Liebermann, D. A., and Fornace, A. J., Jr. (1994) The gadd and MyD genes define a novel set of mammalian genes encoding acidic proteins that synergistically suppress cell growth. Mol. Cell. Biol. 14, 2361–2371 2. Fornace, A. J., Jr., Nebert, D. W., Hollander, M. C., Luethy, J. D., Papathanasiou, M., Fargnoli, J., and Holbrook, N. J. (1989) Mammalian genes coordinately regulated by growth arrest signals and DNA-damaging agents. Mol. Cell. Biol. 9, 4196–4203 3. Hollander, M. C., Sheikh, M. S., Bulavin, D. V., Lundgren, K., Augeri-Henmueller, L., Shehee, R., Molinaro, T. A., Kim, K. E., and Tolosa, E. Ashwell. J. D., Rosenberg, M. P., Zhan, Q., Fernandez-Salguero, P. M., Morgan, W. F., Deng, C. X., and Fornace, A. J. Jr. (1999) Genomic instability in Gadd45a-deficient mice Nat. Genet. 23 176–184 4. Chin, P. L., Momand, J., and Pfeifer, G. P. (1997) In vivo evidence for binding of p53 to consensus binding sites in the p21 and GADD45 genes in response to ionizing radiation. Oncogene 15, 87–99 5. Oyadomari, S., Takeda, K., Takiguchi, M., Gotoh, T., Matsumoto, M., Wada, I., Akira, S., Araki, E., and Mori, M. Nitric oxide-induced apoptosis in pancreatic beta cells is mediated by the endoplasmic reticulum stress pathway. (2001) Proc. Natl. Acad. Sci. USA 98, 10845–10850 6. Oyadomari, S., Araki, E., and Mori, M. (2002) Endoplasmic reticulum stress-mediated apoptosis in pancreatic beta-cells. Apoptosis 7, 335–345 7. Zinszner, H., Kuroda, M., Wang, X., Batchvarova, N., Lightfoot, R. T., Remotti, H., Stevens, J. L., and Ron, D. (1998) CHOP is implicated in programmed cell death in response to impaired function of the endoplasmic reticulum. Genes Dev. 12, 982–995 8. Novoa, I., Zeng, H., Harding, H. P., and Ron, D. (2001) Feedback inhibition of the unfolded protein response by GADD34-mediated dephosphorylation of eIF2alpha. J. Cell Biol. 153, 1011–1022 9. Mengesdorf, T., Althausen, S., Oberndorfer, I., and Paschen, W. (2001) Response of neurons to an irreversible inhibition of endoplasmic reticulum Ca(2+)-ATPase: relationship between global protein synthesis and expression and translation of individual genes. Biochem. J. 356, 805–812
10. Chou, J., and He, B. (1994) Herpes simplex virus 1 gamma(1)34.5 gene function, which blocks the host response to infection, maps in the homologous domain of the genes expressed during growth arrest and DNA damage. Proc. Natl. Acad. Sci. USA 91, 5247–5251 11. He, B., Gross, M., and Roizman, B. (1998) The γ134.5 protein of herpes simplex virus 1 has the structural and functional attributes of a protein phosphatase 1 regulatory subunit and is present in a high molecular weight complex with the enzyme in infected cells. J. Biol. Chem. 273, 20737–20743 12. Adler, H. T., Chinery, R., Wu, D. Y., Kussick, S. J., Payne, J. M., Fornace, A. J., Jr., and Tkachuk, D. C. (1999) Leukemic HRX fusion proteins inhibit GADD34-induced apoptosis and associate with the GADD34 and hSNF5/INI1 proteins. Mol. Cell. Biol. 19, 7050–7060 13. Grishin, A. V., Azhipa, O., Semenov, I., and Corey, S. J. (2001) Interaction between growth arrest-DNA damage protein 34 and Src kinase Lyn negatively regulates genotoxic apoptosis. Proc. Natl. Acad. Sci. USA 28, 10172–10177 14. Brown, S. M., MacLean, A. R., McKie, E. A., and Harland, J. (1997) The herpes simplex virus virulence factor ICP34.5 and the cellular protein MyD116 complex with proliferating cell nuclear antigen through the 63-amino-acid domain conserved in ICP34.5, MyD116, and GADD34. J. Virol. 71, 9442–9449 15. Hasegawa, T., Xiao, H., and Isobe, K. (1999) Biochem. Biophys. Cloning of a GADD34-like gene that interacts with the zinc-finger transcription factor which binds to the p21(WAF) promoter. Res. Commun. 256, 249–254 16. Lord, K. A., Hoffman-Liebermann, B., and Liebermann, D. A. (1990) Sequence of MyD116 cDNA: a novel myeloid differentiation primary response gene induced by IL6. Nucleic Acids Res. 11, 2823 17. Fornace, A. J., Alamo, I., and Hollander, C. (1988) DNA damage-inducible transcripts in mammalian cells. Proc. Natl. Acad. Sci. USA 85, 8800–8804 18. Takeuchi, A., Isobe, K., Miyaishi, O., Sawada, M., Fan, Z., Nakashima, I., and Kiuchi, K. (1998) Microglial NO induces delayed neuronal death following acute injury in the striatum. Eur. J. Neurosci. 10, 1613–1620 19. Hasegawa, T., Xiao, H., and Isobe, K. (1999) Cloning of a GADD34-like gene that interacts with the zinc-finger transcription factor which binds to the p21(WAF) promoter. Biochem. Biophys. Res. Commun. 256, 249–254 20. Wu, S., Hu, Y., Wang, J., Chatterjee, M., Shi, Y., and Kaufman, R. J. (2002) Ultraviolet light inhibits translation through activation of the unfolded protein response kinase PERK in the lumen of the endoplasmic reticulum. J. Biol. Chem. 277, 18077–18083 21. Hollander, M., Zhan, Q., Bae, I., and Fornace, A. J. (1997) Mammalian GADD34, an apoptosis- and DNA damage-inducible gene. J. Biol. Chem. 272, 13731–13737
22. He, B., Gross, M., and Roizman, B. (1997) The gamma(1)34.5 protein of herpes simplex virus 1 complexes with protein phosphatase 1alpha to dephosphorylate the alpha subunit of the eukaryotic translation initiation factor 2 and preclude the shutoff of protein synthesis by double-stranded RNA-activated protein kinase. Proc. Natl. Acad. Sci. USA 94, 843–848 23. Connor, J. H., Weiser, D. C., Li, S., Hallenbeck, J. M., and Shenolikar, S. (2001) Growth arrest and DNA damage-inducible protein GADD34 assembles a novel signaling complex containing protein phosphatase 1 and inhibitor 1. Mol. Cell. Biol. 21, 6841–6850 24. Kaufman, R. J., Scheuner, D., and Schroder, M. (2002) The unfolded protein response in nutrient sensing and differentiation. Nature. 3 411-421. 25. Kaufman, R. J. (1999) Stress signaling from the lumen of the endoplasmic reticulum: coordination of gene transcriptional and translational controls. Genes Dev. 13, 1211–1233 26. Scheuner, D., Song, B., McEwen, E., Liu, C., Laybutt, R., Gillespie, P., Saunders, T., Bonner-Weir, S., and Kaufman, R. J. (2001) Translational control is required for the unfolded protein response and in vivo glucose homeostasis. Mol. Cell 6, 1165–1176 27. Ma, Y., Brewer, J. W., Diehl, J. A., and Hendershot, L. M. (2002) Two distinct stress signaling pathways converge upon the CHOP promoter during the mammalian unfolded protein response. J. Mol. Biol. 318, 1351–1365 28. Takeuchi, A., Mishina, Y., Miyaishi, O., Kojima, E., Hasegawa, T., and Isobe, K. (2003) Heterozygosity with respect to Zfp148 causes complete loss of fetal germ cells during mouse embryogenesis. Nat. Genet., 33, 172–176 29. He, B., Chou, J., Liebermann, D., Hoffman, B., and Rozman, B. (1996) The carboxyl terminus of the murine MyD116 gene substitutes for the corresponding domain of the gamma(1)34.5 gene of herpes simplex virus to preclude the premature shutoff of total protein synthesis in infected human cells. J. Virol. 70, 84–90 30. Berlanga, J. J., Santoyo, J., and De Haro, C. (1999) Characterization of a mammalian homologue of the GCN2 eukaryotic initiation factor 2alpha kinase. Eur. J. Biochem. 265, 754–762 31. Williams, B. R. (1999) PKR; a sentinel kinase for cellular stress. Oncogene 18, 6112–6120 32. Gale, M., Jr., and Katze, M. G. (1998) Molecular mechanisms of interferon resistance mediated by viral-directed inhibition of PKR, the interferon-induced protein kinase. Pharmacol. Ther. 78, 29–46 33. Shi, Y., Vattem, K. M., Sood, R., An, J., Liang, J., Stramm, L., and Wek, R. C. (1998) Identification and characterization of pancreatic eukaryotic initiation factor 2 alpha-subunit kinase, PEK, involved in translational control. Mol. Cell. Biol. 18, 7499–7509
34. Harding, H. P., Zhang, Y., and Ron, D. (1999) Protein translation and folding are coupled by an endoplasmic-reticulum-resident kinase. Nature 397, 271–274 35. Chefalo, P. J., Oh, J., Rafie-Kolpin, M., Kan, B., and Chen, J. J. (1998) Heme-regulated eIF-2alpha kinase purifies as a hemoprotein. Eur. J. Biochem. 258, 820–830 36. Imai, H., Harland, J., McCulloch, J., Graham, D. I., Brown, S. M., and Macrae, I. M. (2002) Specific expression of the cell cycle regulation proteins, GADD34 and PCNA, in the peri-infarct zone after focal cerebral ischaemia in the rat. Eur. J. Neurosci. 15, 1929–1936 37. Talloczy, Z., Jiang, W., Virgin, H. W., Leib, D. A., Scheuner, D., Kaufman, R. J., Eskelinen, E., and Levine, B. (2002) Regulation of starvation- and virus-induced autophagy by the eIF2alpha kinase signaling pathway. Proc. Natl. Acad. Sci. USA 99, 190–195 Received January 30, 2003; accepted April 22, 2003.
Fig. 1
Figure 1. Expression of GADD34 mRNA in vivo. a) Northern blot analysis of GADD34 mRNA expression in fetal stages. Total RNA was extracted from C57BL/6 mouse embryo at the indicated developmental stage from E8.5 to E18.5; 20 µg of total RNA was electrophoresed and hybridized with 32P-labeled GADD34 cDNA probe. Human 28S rRNA probe was used as an internal RNA-loading control. These experiments were performed more than three times, and all showed same expression patterns. b) In situ hybridization of E12.5 embryos. Expression of GADD34 mRNA on E12.5 was detected by using anti-sense (left) and sense (right) primer (original magnification, 40×). c) Northern blot analysis of GADD34 mRNA expression of tissues in adult mice. Total RNA (20 µg) extracted from 2-month-old C57BL/6 mouse tissues was eletrophoresed and hybridized with 32P-labeled GADD34 cDNA probe.
Fig. 2
Figure 2. Targeting of GADD34. a) Design of the GADD34 targeting constructs. “fw”, “wild”, and “neo” are PCR primers for genotyping. b) PCR genotyping of mouse tail DNA. c) Western blot analysis of GADD34 in MMS-treated MEFs (GADD34+/+, GADD34+/–, GADD34–/–) and in NIH3T3 cells, which were transfected with GADD34 cDNA (pcDNA3.1 Myc-His vector containing a murine GADD34 cDNA or empty pcDNA3.1 Myc-His vector). Total proteins were prepared at 6 h after MMS treatment. Cell extracts were analyzed by using an anti-rabbit GADD34 antibody. UT; untreated control. d) Two-month-old GADD34+/+, GADD34+/–, and GADD34–/– mice were intraperitoneally injected with 1.5 mmol/kg of MMS and killed at the times indicated at the top of each lane. About 100 µg of liver protein extracts were electrophoresed on SDS/PAGE gel, blotted, and probed with GADD34 antibody.
Fig. 3
Figure 3. ER stresses (tunicamycin, Tm; thapsigargin, Tg or DTT) induce GADD34 expression. MEFs were treated with 0–10 µg/ml of Tm, 0–1.0 µM of Tg, 0–10 mM of DTT, or 0–80 µg/ml of MMS. In the case of Tg and DTT treatment, MEFs were washed off and changed to fresh medium at 0.5 h. Shown are immunoblots with a rabbit polyclonal antibody against GADD34. a) Dose-dependent GADD34 protein expression at 6 h post-treatment (b) time-dependent GADD34 protein expression. MEFs were exposed to 5 µg/ml of Tm or 80 µg/ml of MMS for the indicated period of time. MEFs were also exposed to 1.0 µM of Tg or 10 mM of DTT for 0.5 h, followed by different recovery times. UT; untreated control.
Fig. 4
Figure 4. Phosphorylation of eIF2α-induced by Tg or DTT is reversed by GADD34. GADD34+/+ MEFs or GADD34 MEFs were treated with 1 µM of Tg (a) or 10 mM of DTT (b), washed off and changed to fresh medium at 0.5 h. Total cell extracts were taken from these cells at different times after treatment. Shown are immunoblots with a rabbit polyclonal antibody against GADD34 and eIF2α phosphorylated on Ser51, and a goat polyclonal antibody that recognizes both phosphorylated and unphosphorylated eIF2α.
–/–
Fig. 5
Figure 5. Tg-induced eIF2α phosphorylation levels in GADD34–/– MEFs were reduced by overexpression of GADD34. GADD34–/– MEFs were transfected with either empty pcDNA3.1 Myc-His vector or pcDNA3.1 Myc-His vector containing a murine GADD34 cDNA. Cells were treated with 1 µM of Tg and changed to fresh medium at 0.5 h. Whole cell lysates were harvested at the indicated times. Shown are immunoblots with a rabbit polyclonal antibody against GADD34, a rabbit polyclonal antibody against eIF2αphosphorylated on Ser51, and a goat polyclonal antibody that recognizes both phosphorylated and unphosphorylated eIF2α .
Fig. 6
Figure 6. Protein synthesis after Tg treatment in GADD34+/+, GADD34+/–, and GADD34–/– MEFs. MEFs were exposed to Tg (1 µM) for 0.5 h, followed by different recovery times. After pulse-labeling with [35S] methionine, whole cell lysates (10 µg) were resolved by 12% SDS-PAGE. a) The nascent protein synthesis after Tg treatment was determined by autoradiography (left panel). The right panel shows a Coomassie blue stain of the same gel to confirm the equal amount of total proteins in each lane. These results are representative of the four separate experiments using different pairs of MEFs. b) The nascent protein synthesis after DTT treatment was determined by autoradiography.
Fig. 7
Figure 7. Expressions of ATF4, BiP (GRP78) and CHOP (GADD153) induced by Tg treatment are decreased in GADD34–/– MEF. MEFs were treated with 1 µM of Tg for 0.5 h. Total cell extracts were taken from these cells at different times after treatment. a) Immunoblot of ATF4, BiP, or CHOP in Tg-treated GADD34+/+ MEFs or GADD34–/– MEFs. b) Northern blot of RNA from Tg-treated GADD34+/+ MEFs or GADD34–/– MEFs. Mouse 28S rRNA probe was used as an internal RNA-loading control.
