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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online June 3, 2003 as doi:10.1096/fj.02-1184fje. |
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* Department of Basic Gerontology, National Institute for Longevity Sciences, Morioka-cho, Obu, Aichi 474-8522, Japan;
Department of Internal Medicine, Nagoya University Graduate School of Medicine, Showa-ku, Nagoya, Aichi 466-8520, Japan; and
Department of Host Defense, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
2Correspondence: Department of Basic Gerontology, National Institute for Longevity Sciences, 36-3, Gengo, Morioka-cho, Obu, Aichi 474-8522, Japan. E-mail: kenisobe{at}nils.go.jp
SPECIFIC AIMS
GADD34 is a member of the protein family whose expression is up-regulated by growth arrest and DNA damage, but its function has not yet been elucidated in vivo. We tried to find the function of GADD34 by constructing GADD34-deficient mice.
PRINCIPAL FINDINGS
1. GADD34 is highly expressed at the fetal stage and in adult mouse organs
At the fetal stage of C57BL/6CR mice, GADD34 mRNA expression was detected on embryonic day 8.5 (E8.5), E9.5, E12.5 (especially strong), E14.5, and E18.5. Almost no expression was detected on E10.5, E11.5, and E13.5. The distribution of GADD34 was ubiquitously expressed on E12.5; it is expressed at high levels in the brain, spinal cord, tongue, lung, and genital tubercle. 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).
2. GADD34 knockout mice show normal development and normal fertility
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 (Stratagene, San Diego, CA, USA). 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 did not show abnormal phenotypes or signs of disease during the first 12 months of life.
3. GADD34 is strongly induced in response to endoplasmic reticulum (ER) stress
Previous studies demonstrated that rodent GADD34 was inducible by treatment of cell lines with various DNA-damaging agents such as alkylating agent and that it was mediated by some of the agents causing unfolded protein response (UPR). Tunicamycin (Tm) inhibits asparagines (N) -linked glycosylation and dithiothreitol (DTT) disrupts disulfide bond formation. Thapsigargin (Tg) is an irreversible 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 >0.05 µM and 5 mM, respectively, with levels remaining unchanged. All three ER stress-inducible agents elevated the level of GADD34 in a time-dependent manner.
4. GADD34 is required for eukaryotic translation initiation factor 2
(eIF2) dephosphorylation and recovery from a shutoff of total protein synthesis in response to ER stress
The COOH terminus of GADD34 is homologous to the corresponding domain of the herpes simplex virus-encoded protein 
134.5. Both 134.5 and GADD34 interact with PP1 and dephosphorylate eIF2. To determine whether the expression of GADD34 affects eIF2 phosphorylation levels, we compared those levels of GADD34+/+ MEFs treated with ER stress-inducing agent with those of GADD34-/- MEFs. In wild-type MEFs, the eIF2 phosphorylation level was temporarily increased just after treatment with Tg or DTT and promptly decreased, whereas in GADD34-deficient MEFs eIF2 is strongly phosphorylated and sustained a high level of phosphorylation (Fig. 1
a, b). By contrast, after treatment with tunicamycin, the eIF2 phosphorylation level did not change in either GADD34+/+ or -/- cells. Phosphorylation of eIF2 reduces its functional level and limits initiation translation on all cellular mRNAs within the cells. To clarify whether strongly phosphorylated eIF2 in Tg- or DTT-treated GADD34-/- MEFs affects translation, we examined the new protein synthesis rates in the treated MEFs. In both genotypes of MEFs, protein synthesis was severely reduced just after Tg treatment. Protein synthesis gradually recovered in GADD34+/+ MEFs but remained at lower levels even after 12 h of Tg treatment in GADD34-/- MEFs (Fig. 2
a). By treatment with another ER stress agent, DTT, GADD34-/- cells also kept protein synthesis at a low level until 2 h after treatment (Fig. 2b
). These results indicate that highly phosphorylated eIF2 in GADD34-/- MEFs leads to a reduction of newly synthesized protein.
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5. GADD34 affects expression of BiP/GRP78 (binding Ig protein/glucose regulated protein of molecular weight 78 kDa) and CHOP (C/EBP homologous protein)/GADD153 in response to ER stress
Bip/GRP78 and CHOP/GADD153 have been shown to be downstream target proteins in ER stress responses. Bip, a classical marker for UPR activation, acts as a chaperone in the UPR. CHOP has been reported as an ER stress-associated apoptosis factor. In the case of ER stress, eIF2 are phosphorylated and subsequently ATF4 (activating transcription factor 4) induced by phosphorylated eIF2. Transcription of Bip and CHOP are both activated by ATF4 through binding their amino acid response element. We analyzed ATF4, Bip, and CHOP expressions in GADD34-deficient MEF under ER stress. Although ATF4, Bip, and CHOP are strongly induced by Tg treatment in wild-type MEF, they are weakly induced by Tg treatment in GADD34-deficient MEF. This indicates that GADD34 exists upstream of ATF4, BiP, and CHOP and regulates their expressions in the UPR.
CONCLUSIONS
We have shown here that GADD34 was expressed in normal tissues in an unstimulated state and that its expression in the fetus was unexpected. We showed that at the fetal stage, GADD34 expression was especially strong on E12.5. Almost no expression was detected on E10.5, E11.5, and E13.5. It is of interest to know why GADD34 expression was different in the fetal stages. Despite the stage-specific expression of GADD34, mice deficient in it had no abnormalities under normal breeding conditions. Other proteins with functions similar 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 eIF2 induced by Tg or DTT. These treatments, which induce an ER stress response, shut off the protein synthesis of both GADD34-deficient and wild-type MEF. However, in wild-type MEF an early recovery from the shutoff is observed that correlates with GADD34 expression. Despite the agent that disrupts protein folding in the ER, in the present study Tm induced neither phosphorylation of eIF2 nor shutoff of the protein synthesis. This result 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 diminished the phosphorylation of eIF2 levels in response to ER stress. GADD34 formed a complex with the catalytic subunit of protein phosphatase 1 (PP1) that specifically promoted the dephosphorylation of eIF2 These results correlate with the function of 134.5 of herpes simplex virus (HSV), which is found to combine with PP1 and dephosphorylates eIF2, thereby precluding the shutoff of protein synthesis. It has been reported that transcriptional activation of CHOP and BiP upon activation of the UPR requires eIF2 phosphorylation. We have shown here that transcriptional and translational expression of CHOP and BiP are both strongly induced by ER stresses in wild-type MEF, presumably through ATF4. On the other hand, this induction is weak in GADD34-deficient MEF despite sustaining a high level of phosphorylated eIF2. These results suggest that a GADD34-related pathway of CHOP and BiP expression in response to ER stress exists regardless of the eIF2 phosphorylation level (Fig. 3
).
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Although translational attenuation caused by ER stress has been discussed in great detail, a recovery from shutoff of protein synthesis has not been extensively studied. 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 in maintaining homeostasis of cells in the UPR. Recent work provided us with possible roles for the recovery from a shutoff of protein synthesis caused by GADD34 in vivo. It was reported recently that GADD34 is expressed in the peri-infarct zone after focal cerebral ischemia in rats. It has also been reported that mammalian eIF2 kinase-dependent autophagy is antagonized by the HSV-encoded neurovirulence gene product ICP34.5. Further analysis of the GADD34 engaged molecular pathway of the recovery from a shutoff of protein synthesis will open a new field in the pathophysiology of the mammalian system.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-1184fje; doi: 10.1096/fj.02-1184fje 
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