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. 2005 Mar 23;24(6):1243-55.
doi: 10.1038/sj.emboj.7600596. Epub 2005 Mar 10.

TRB3, a novel ER stress-inducible gene, is induced via ATF4-CHOP pathway and is involved in cell death

Nobumichi Ohoka  1 , Satoshi Yoshii, Takayuki Hattori, Kikuo Onozaki, Hidetoshi Hayashi
Affiliations

TRB3, a novel ER stress-inducible gene, is induced via ATF4-CHOP pathway and is involved in cell death

Nobumichi Ohoka et al. EMBO J. .

Abstract

C/EBP homologous protein (CHOP) is a stress-inducible nuclear protein that is crucial for the development of programmed cell death and regeneration; however, the regulation of its function has not been well characterized. Slbo, a Drosophila homolog of C/EBP (CCAAT/enhancer binding protein), was shown to be unstabilized by tribbles. Here, we identified TRB3 as a tribbles ortholog in humans, which associated with CHOP to suppress the CHOP-dependent transactivation. TRB3 is induced by various forms endoplasmic reticulum (ER) stress later than CHOP. Tunicamycin treatment enhanced the TRB3 promoter activity, while dominant-negative forms of CHOP suppressed the tunicamycin-induced activation. In addition, the tunicamycin response region in the TRB3 promoter contains amino-acid response elements overlapping the CHOP-binding site, and CHOP and ATF4 cooperated to activate this promoter activity. Knockdown of endogenous ATF4 or CHOP expression dramatically repressed tunicamycin-induced TRB3 induction. Furthermore, knockdown of TRB3 expression decreased ER stress-dependent cell death. These results indicate that TRB3 is a novel target of CHOP/ATF4 and downregulates its own induction by repression of CHOP/ATF4 functions, and that it is involved in CHOP-dependent cell death during ER stress.

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Figures

Figure 1
Figure 1
TRB3 is induced during ER stress. (A, B) 293 (A) or HepG2 cells (B) were treated with 2 μg/ml of tunicamycin in the presence or absence of 10 μg/ml of cycloheximide (CHX) for the indicated periods. Total RNA was prepared and analyzed by Northern blotting using each specific probe. (C) 293 cells were treated with 2 μg/ml of tunicamycin, 10 μM MG132, 100 μg/ml of MMS or 2 μM A23187 for 6 h. Each mRNA level in the cells was analyzed by RT–PCR using specific primers. TRB3, 32 cycles ; CHOP, 30 cycles ; GAPDH, 24 cycles. (D) 293 (top), A375 (middle) and HepG2 (bottom) cells were treated with 2 μg/ml of tunicamycin for the indicated periods. Cell lysate was prepared and equal amounts of protein were subjected to SDS–PAGE. Western blotting was performed with anti-human TRB3, anti-CHOP, anti-ATF4 or anti-β-actin antibodies.
Figure 2
Figure 2
TRB3 interacts with CHOP in vivo. (A) Constructs of CHOP mutants. (B, D) 293 cells were transiently transfected with the indicated constructs. After 36 h, cells were treated with 10 μM MG132 for 12 h. The cell lysates were immunoprecipitated with anti-Flag antibody and immunoblotted with anti-Myc antibody. The expression level of each protein was assessed by the immunoblotting of cell lysates with anti-Flag or anti-Myc antibodies. (C) Deletion mutants of TRB3. KD: kinase-like domain.
Figure 3
Figure 3
TRB3 represses CHOP transactivation. (A) Alignment of CHOP-responsive element in various genes. (B) 293 cells were transiently transfected with the expression plasmids for wild-type or deletion mutants of CHOP fused with GAL4-dbd, pCMV5-Gal4-CHOPs, pFR-luc and pCMV-β-gal. After 48 h, the luciferase activity in cell lysates was measured and was normalized with β-galactosidase activity. (C, D) A375 cells were transiently transfected with p(CHOP)4-Luc, pCMV-β-gal and various combinations of the expression vectors for CHOP and NF-IL6 with or without TRB3. After 48 h, the luciferase activity in cell lysates was measured and was normalized with β-galactosidase activity. (E) 293 cells were transiently transfected with the expression plasmids for Gal4-CHOP(WT) and/or TRB3, pFR-luc and pCMV-β-gal. After 48 h, the luciferase activity in cell lysates was measured and was normalized with β-galactosidase activity. The inset depicts the enlarged figure for the data of GAL4-dbd. (F, G) A375 cells were transiently transfected with p(CHOP)4-Luc, pCMV-β-gal and various combinations of the expression vectors for CHOP and NF-IL6 with or without wild type or deletion mutants of TRB3. After 48 h, the luciferase activity in cell lysates was measured and was normalized with β-galactosidase activity. Similar results were obtained in three independent experiments.
Figure 4
Figure 4
Overexpression of CHOP causes TRB3 induction. (A) 293 cells were transiently transfected with pTRB3-Luc and pCMV-β-gal. After 24 h, cells were left untreated or treated with 2 μg/ml of tunicamycin for 16 h. The luciferase activity in cell lysates was measured and was normalized with β-galactosidase activity. (B) 293 cells were transiently transfected with pTRB3-Luc and pCMV-β-gal combination with expression vectors for CHOP and/or NF-IL6 for 48 h. The luciferase activity in cell lysates was measured and was normalized with β-galactosidase activity. (C) 293 cells were transiently transfected with pTRB3-Luc and pCMV-β-gal in the presence of expression vectors for wild type or mutants of CHOP or TRB3. After 24 h, cells were left untreated or treated with 2 μg/ml of tunicamycin for 16 h. The luciferase activity in cell lysates was measured and was normalized with β-galactosidase activity. (D) 293 cells were transiently transfected with pTRB3-Luc, pCMV-β-gal, control siRNA and/or TRB3 siRNA. After 36 h, cells were left untreated or treated with 2 μg/ml of tunicamycin for 16 h. The luciferase activity in cell lysates was measured and was normalized with β-galactosidase activity. Similar results were obtained in three independent experiments.
Figure 5
Figure 5
Mutagenesis analysis of the TRB3 promoter. (A) Constructs of TRB3 promoter reporter genes. Position +1 demonstrates the initiation site for the TRB3 transcription. (B) 293 cells were transiently transfected with pTRB3-Luc, pCMV-β-gal and the expression vectors for CHOP/NF-IL6 or ATF4. After 24 h, cells were left untreated or treated with 2 μg/ml of tunicamycin for 16 h. The luciferase activity in cell lysates was measured and was normalized with β-galactosidase activity. (C) The sequence of the ERSE in the TRB3 promoter. (D) Constructs of TRB3 promoter reporter genes. (E) HepG2 cells were transiently transfected with pTRB3-Luc and pCMV-β-gal, and after 24 h, treated with 2 μg/ml of tunicamycin for 16 h (left). 293 cells were transiently transfected with pTRB3-Luc, pCMV-β-gal and the indicated expression vectors for CHOP/NF-IL6 or ATF4. The luciferase activity in cell lysates was measured and normalized with β-galactosidase activity (right). The results in HepG2 cells were shown because the tunicamycin-induced activation level was higher than in 293 cells. Similar results were obtained in 293 cells as well. Similar results were obtained in three independent experiments.
Figure 6
Figure 6
CHOP and ATF4 cooperate to activate the TRB3 promoter. (A) Alignment of the AARE in the TRB3 promoter with its related elements. (B–E) 293 cells were transiently transfected with pTRB3-Luc and pCMV-β-gal in combination with the indicated expression vectors for 48 h. The luciferase activity in cell lysates was measured and normalized with β-galactosidase activity. Similar results were obtained in three independent experiments.
Figure 7
Figure 7
Knockdown of either ATF4 or CHOP repressed TRB3 induction. (A) Wild-type and NF-IL6−/− MEFs were treated with 2 μg/ml of tunicamycin for the indicated periods. Total RNA was prepared and analyzed by Northern blotting using indicated mouse cDNAs as probes. (B, C) 293 cells were transiently transfected with control siRNA, ATF4 siRNA or CHOP siRNA. After 48 h, cells were treated with 2 μg/ml of tunicamycin for the indicated periods. The cell lysates were analyzed by immunoblotting using anti-TRB3, anti-ATF4, anti-CHOP and anti-β-actin (bottom). Similar results were obtained in three independent experiments.
Figure 8
Figure 8
TRB3 induces ER stress-dependent cell death. (A) 293 cells were transiently transfected with control siRNA or TRB3 siRNA. After 48 h, cells were treated with 2 μg/ml of tunicamycin for the indicated periods. The percentage of adherent cells was measured by crystal violet staining. The right panel shows the stained cells treated with tunicamycin for 48 h. (B, C) HeLa cells (B) were transiently transfected with control siRNA or TRB3 siRNA. 293 cells (C) were transiently transfected with control vector or Flag-TRB3. After 48 h, cells were treated with 2 μg/ml of tunicamycin for 24 h or left untreated in the absence or presence of 50 μM of zVAD. The percentage of dead cells was measured by Trypan blue staining. (D) HeLa cells were transiently transfected with control siRNA or TRB3 siRNA. After 48 h, cells were treated with 2 μg/ml of tunicamycin for 24 h. Apoptotic cells were measured by staining with DAPI. Arrowheads in the right panel indicate apoptotic cells. Similar results were obtained in three independent experiments.
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