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. 2020 Nov 27;40(11):BSR20201289.
doi: 10.1042/BSR20201289.

Mitochondrial translation inhibition triggers ATF4 activation, leading to integrated stress response but not to mitochondrial unfolded protein response

Katsuhiko Sasaki #  1   2   3 , Takeshi Uchiumi #  1   4 , Takahiro Toshima #  1 , Mikako Yagi  1   4 , Yura Do  1 , Haruka Hirai  1   4 , Ko Igami  1   2   3 , Kazuhito Gotoh  1 , Dongchon Kang  1
Affiliations

Mitochondrial translation inhibition triggers ATF4 activation, leading to integrated stress response but not to mitochondrial unfolded protein response

Katsuhiko Sasaki et al. Biosci Rep. .

Abstract

Mitochondrial-nuclear communication, known as retrograde signaling, is important for regulating nuclear gene expression in response to mitochondrial dysfunction. Previously, we have found that p32/C1qbp-deficient mice, which have a mitochondrial translation defect, show endoplasmic reticulum (ER) stress response and integrated stress response (ISR) gene expression in the heart and brain. However, the mechanism by which mitochondrial translation inhibition elicits these responses is not clear. Among the transcription factors that respond to mitochondrial stress, activating transcription factor 4 (ATF4) is a key transcription factor in the ISR. Herein, chloramphenicol (CAP), which inhibits mitochondrial DNA (mtDNA)-encoded protein expression, induced eukaryotic initiation factor 2 α subunit (eIF2α) phosphorylation and ATF4 induction, leading to ISR gene expression. However, the expression of the mitochondrial unfolded protein response (mtUPR) genes, which has been shown in Caenorhabditis elegans, was not induced. Short hairpin RNA-based knockdown of ATF4 markedly inhibited the CAP-induced ISR gene expression. We also observed by ChIP analysis that induced ATF4 bound to the promoter region of several ISR genes, suggesting that mitochondrial translation inhibition induces ISR gene expression through ATF4 activation. In the present study, we showed that mitochondrial translation inhibition induced the ISR through ATF4 activation rather than the mtUPR.

Keywords: ATF4; integratad stress responce; mitochondria; mtUPR.

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Conflict of interest statement

The authors declare that there are no competing interests associated with the manuscript.

Figures

Figure 1
Figure 1. CAP inhibits mitochondrial translation
(A) CAP-mediated inhibition of mitochondrial translation efficiency was monitored by Western blot analysis of the abundance of CoxI and CoxIII proteins encoded by mtDNA. MEFs were incubated with control or 100 μg/ml CAP-containing medium for 24 and 48 h. Immunodetection of GAPDH and SDHA was used as the loading control. All experiments were done in triplicate. Quantification is shown in the right panel. ***P<0.005. (B) Antibiotics such as Chloramphenicol (CAP)- and Doxycycline (DOX)-mediated CoxI expression was monitored by Western blot analysis for 48 h in MEF cells. Immunodetection of β-actin was used as the loading control. (C) In vivo mitochondrial translation was performed for 60 min. The products were labeled during the reaction with a mixture of [35S]-methionine and [35S]-cysteine in the presence of emetine and/or CAP, and then detected by autoradiography after SDS/PAGE (left panel). Deficient translation was observed in CAP-treated MEFs. Equal loading was confirmed by CBB staining (right panel). The proteins indicated are representative proteins based on their molecular weight.
Figure 2
Figure 2. CAP induces the ER stress response
(A) The levels of eIF2α phosphorylation at Ser51 and ATF4 expression were evaluated in MEFs incubated with or without 100 μg/ml CAP for 48 h. The abundance of both phosphorylated and total eIF2α was assessed by Western blotting. Immunodetection of β-actin was used as the loading control. All experiments were done in triplicate. Quantification is shown in the right panel. *P<0.05 **P<0.01, ***P<0.001 vs control. (B) Chop-10 mRNA abundance was evaluated by RT-qPCR in MEFs treated with CAP. Chop-10 mRNA abundance was also evaluated in MEFs treated with the antibiotics, actinonin (50 μM), doxycycline (100 μg/ml) and CAP (100 μg/ml), for 48 h. Chop-10 mRNA was normalized to 18S rRNA, which was the loading control. All experiments were done in triplicate; *P<0.05.
Figure 3
Figure 3. Interfering with mitochondrial translation in wild type MEFs triggers the ISR, but not the mtUPR
(A,B) The mRNA abundance of the ISR-related gene markers, Trib3, Atf3 and Gadd45, in MEFs treated with 50 μM CAP (A) or 50 μM Acti (actinonin) (B) for 48 h was assessed by RT-qPCR. Results are expressed the mean ± SD of three independent experiments. (C,D) The abundance of ClpP, Hsp70, Hsp10 and Hsp60 mRNA in MEFs treated with CAP (C) or Actinonin (D) for 24 and 48 h was assessed by RT-qPCR. Results are expressed as the mean ± SD of three independent experiments. (E) FGF21 and GDF15 gene expression were up-regulated in 6-week-old p32cKO brain compared with control brain. Results are expressed as the mean ± SD of three independent experiments. (F) qRT-PCR analysis confirmed that ISR gene is up-regulated, but not mtUPR marker gene expression in 6-week-old p32cKO brain compared with control brain. Results are expressed the mean ± SD of three independent experiments. *P<0.05 **P<0.01, ***P<0.005 vs. control.
Figure 4
Figure 4. Effect of ATF4 silencing on CAP-induced ISR gene expression
(A) MEFs stably transfected with either non-targeting shRNA (shGFP) or with two different shRNAs against ATF4 (shATF4) for 24 h was replaced with control medium or with medium containing CAP (100 μg/ml) for 24 h. CAP induced CoxI decline were assessed by Western blotting. Immunodetection of GAPDH was used as the loading control. All experiments were done in triplicate. Quantification is shown in the right panel.*P<0.05, ***P<0.005 vs control. (B) MEFs which were stably transfected were incubated with or without CAP (100 μg/ml) for 12 h. The mRNA expression of ISR and mtUPR-related genes was evaluated by RT-qPCR. Results are expressed as fold change to control and are expressed the mean ± SD of three independent experiments. ***P<0.005 vs. control.
Figure 5
Figure 5. ATF4 directly activates ISR gene promoters
(A) ATF4 was immunoprecipitated with anti-ATF4 antibodies after formaldehyde cross-linking of MEFs treated with CAP. We used anti IgG antibody for negative control. (B) Chromatin was immunoprecipitated, followed by quantitation by PCR analysis of the region containing the CARE site in ISR gene promoters (Fgf21, Gadd45, Sestrin2 Cdsn, Trib3, Atf3, Atf6). Il-6 exon 2 was used as a negative control. Results are expressed as the mean ± SD of three independent experiments.
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