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A sustained deficiency of mitochondrial respiratory complex III induces an apoptotic cell death through the p53-mediated inhibition of pro-survival activities of the activating transcription factor 4.

Evstafieva AG, Garaeva AA, Khutornenko AA, Klepikova AV, Logacheva MD, Penin AA, Novakovsky GE, Kovaleva IE, Chumakov PM - Cell Death Dis (2014)

Bottom Line: We found that a short-term (5 h) inhibition of ETC complex III with myxothiazol results in the phosphorylation of translation initiation factor eIF2α and upregulation of mRNA for the activating transcription factor 4 (ATF4) and several ATF4-regulated genes.Interestingly, a sustained inhibition of ETC complex I by piericidine did not induce the p53 response and stably maintained the pro-survival activation of ATF4/ISR.We conclude that a downregulation of mitochondrial ETC generally induces adaptive pro-survival responses, which are specifically abrogated by the suicidal p53 response triggered by the genetic risks of the pyrimidine nucleotide deficiency.

View Article: PubMed Central - PubMed

Affiliation: 1] Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, 119992, Russia [2] Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University, Moscow, 119992, Russia.

ABSTRACT
Generation of energy in mitochondria is subjected to physiological regulation at many levels, and its malfunction may result in mitochondrial diseases. Mitochondrial dysfunction is associated with different environmental influences or certain genetic conditions, and can be artificially induced by inhibitors acting at different steps of the mitochondrial electron transport chain (ETC). We found that a short-term (5 h) inhibition of ETC complex III with myxothiazol results in the phosphorylation of translation initiation factor eIF2α and upregulation of mRNA for the activating transcription factor 4 (ATF4) and several ATF4-regulated genes. The changes are characteristic for the adaptive integrated stress response (ISR), which is known to be triggered by unfolded proteins, nutrient and metabolic deficiency, and mitochondrial dysfunctions. However, after a prolonged incubation with myxothiazol (13-17 h), levels of ATF4 mRNA and ATF4-regulated transcripts were found substantially suppressed. The suppression was dependent on the p53 response, which is triggered by the impairment of the complex III-dependent de novo biosynthesis of pyrimidines by mitochondrial dihydroorotate dehydrogenase. The initial adaptive induction of ATF4/ISR acted to promote viability of cells by attenuating apoptosis. In contrast, the induction of p53 upon a sustained inhibition of ETC complex III produced a pro-apoptotic effect, which was additionally stimulated by the p53-mediated abrogation of the pro-survival activities of the ISR. Interestingly, a sustained inhibition of ETC complex I by piericidine did not induce the p53 response and stably maintained the pro-survival activation of ATF4/ISR. We conclude that a downregulation of mitochondrial ETC generally induces adaptive pro-survival responses, which are specifically abrogated by the suicidal p53 response triggered by the genetic risks of the pyrimidine nucleotide deficiency.

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The time-dependent effect of mitochondrial ETC complex III inhibition on the expression of ATF4 and its target genes in HCT116 cells. (a–f) The effects of myxothiazol (1 μM) for indicated intervals of time on ATF4, CHOP, TRIB3, SLC7A11, CHAC1 and TP53INP1 mRNA levels in HCT116 cells were examined by RT-qPCR. Mean and S.D. are presented of three independent experiments. All values are normalized to the level of the corresponding mRNA in the control (untreated) cells. (g) Western analysis of p53 in myxothiazol-treated HCT116 cells for indicated intervals of time
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fig2: The time-dependent effect of mitochondrial ETC complex III inhibition on the expression of ATF4 and its target genes in HCT116 cells. (a–f) The effects of myxothiazol (1 μM) for indicated intervals of time on ATF4, CHOP, TRIB3, SLC7A11, CHAC1 and TP53INP1 mRNA levels in HCT116 cells were examined by RT-qPCR. Mean and S.D. are presented of three independent experiments. All values are normalized to the level of the corresponding mRNA in the control (untreated) cells. (g) Western analysis of p53 in myxothiazol-treated HCT116 cells for indicated intervals of time

Mentions: Surprisingly, at the later time points of complex III inhibition (13–17 h), the expression of ATF4 mRNA switched from a 2.5-fold upregulation to a 2.3- and 4-fold downregulation (Table 1). The expression of the above ATF4 target genes (except ASNS) either returned to control levels (CHOP) or even dropped further below (CHAC1, SLC7A11, TRIB3). After 17 h of myxothiazol treatment, the upregulated ASNS mRNA levels also decreased from 3.6-fold at 5 h to 1.5-fold. Apparently, the changes observed following ETC inhibition could not be explained simply by the UPR/ISR gene expression program. To clarify this point, the effect of complex III inhibition on ATF4 mRNA levels at different time points was examined by RT-qPCR analysis. We observed a time-dependent change in ATF4 mRNA level in HCT116 (Figure 2a), RKO (Supplementary Figure S1a) and HeLa (Supplementary Figure S2a) cell lines. Following myxothiazol treatment, the levels of ATF4 mRNA were increased at an early time point (4 h) and then dropped below the control level (16 h). A similar time dependence (but sometimes shifted toward later time points) was obtained for the selected ATF4 transcriptional targets DDIT3/CHOP, TRIB3, SLC7A11 and CHAC1 (Figures 2 b–d, f; Supplementary Figure S1 b–d, f; Supplementary Figure S2 b–d). Therefore, the RT-qPCR analysis has confirmed the mRNA- seq results and has shown that the suppression of ATF4 in response to a sustained ETC complex III inhibition was not cell-line specific. Besides, western analysis has shown that following myxothiazol treatment, the ATF4 protein levels were also increased at early time points (3–5 h) and then dropped to (16 h) or below (24 h) the control levels (Supplementary Figure S1h).


A sustained deficiency of mitochondrial respiratory complex III induces an apoptotic cell death through the p53-mediated inhibition of pro-survival activities of the activating transcription factor 4.

Evstafieva AG, Garaeva AA, Khutornenko AA, Klepikova AV, Logacheva MD, Penin AA, Novakovsky GE, Kovaleva IE, Chumakov PM - Cell Death Dis (2014)

The time-dependent effect of mitochondrial ETC complex III inhibition on the expression of ATF4 and its target genes in HCT116 cells. (a–f) The effects of myxothiazol (1 μM) for indicated intervals of time on ATF4, CHOP, TRIB3, SLC7A11, CHAC1 and TP53INP1 mRNA levels in HCT116 cells were examined by RT-qPCR. Mean and S.D. are presented of three independent experiments. All values are normalized to the level of the corresponding mRNA in the control (untreated) cells. (g) Western analysis of p53 in myxothiazol-treated HCT116 cells for indicated intervals of time
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4260727&req=5

fig2: The time-dependent effect of mitochondrial ETC complex III inhibition on the expression of ATF4 and its target genes in HCT116 cells. (a–f) The effects of myxothiazol (1 μM) for indicated intervals of time on ATF4, CHOP, TRIB3, SLC7A11, CHAC1 and TP53INP1 mRNA levels in HCT116 cells were examined by RT-qPCR. Mean and S.D. are presented of three independent experiments. All values are normalized to the level of the corresponding mRNA in the control (untreated) cells. (g) Western analysis of p53 in myxothiazol-treated HCT116 cells for indicated intervals of time
Mentions: Surprisingly, at the later time points of complex III inhibition (13–17 h), the expression of ATF4 mRNA switched from a 2.5-fold upregulation to a 2.3- and 4-fold downregulation (Table 1). The expression of the above ATF4 target genes (except ASNS) either returned to control levels (CHOP) or even dropped further below (CHAC1, SLC7A11, TRIB3). After 17 h of myxothiazol treatment, the upregulated ASNS mRNA levels also decreased from 3.6-fold at 5 h to 1.5-fold. Apparently, the changes observed following ETC inhibition could not be explained simply by the UPR/ISR gene expression program. To clarify this point, the effect of complex III inhibition on ATF4 mRNA levels at different time points was examined by RT-qPCR analysis. We observed a time-dependent change in ATF4 mRNA level in HCT116 (Figure 2a), RKO (Supplementary Figure S1a) and HeLa (Supplementary Figure S2a) cell lines. Following myxothiazol treatment, the levels of ATF4 mRNA were increased at an early time point (4 h) and then dropped below the control level (16 h). A similar time dependence (but sometimes shifted toward later time points) was obtained for the selected ATF4 transcriptional targets DDIT3/CHOP, TRIB3, SLC7A11 and CHAC1 (Figures 2 b–d, f; Supplementary Figure S1 b–d, f; Supplementary Figure S2 b–d). Therefore, the RT-qPCR analysis has confirmed the mRNA- seq results and has shown that the suppression of ATF4 in response to a sustained ETC complex III inhibition was not cell-line specific. Besides, western analysis has shown that following myxothiazol treatment, the ATF4 protein levels were also increased at early time points (3–5 h) and then dropped to (16 h) or below (24 h) the control levels (Supplementary Figure S1h).

Bottom Line: We found that a short-term (5 h) inhibition of ETC complex III with myxothiazol results in the phosphorylation of translation initiation factor eIF2α and upregulation of mRNA for the activating transcription factor 4 (ATF4) and several ATF4-regulated genes.Interestingly, a sustained inhibition of ETC complex I by piericidine did not induce the p53 response and stably maintained the pro-survival activation of ATF4/ISR.We conclude that a downregulation of mitochondrial ETC generally induces adaptive pro-survival responses, which are specifically abrogated by the suicidal p53 response triggered by the genetic risks of the pyrimidine nucleotide deficiency.

View Article: PubMed Central - PubMed

Affiliation: 1] Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, 119992, Russia [2] Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University, Moscow, 119992, Russia.

ABSTRACT
Generation of energy in mitochondria is subjected to physiological regulation at many levels, and its malfunction may result in mitochondrial diseases. Mitochondrial dysfunction is associated with different environmental influences or certain genetic conditions, and can be artificially induced by inhibitors acting at different steps of the mitochondrial electron transport chain (ETC). We found that a short-term (5 h) inhibition of ETC complex III with myxothiazol results in the phosphorylation of translation initiation factor eIF2α and upregulation of mRNA for the activating transcription factor 4 (ATF4) and several ATF4-regulated genes. The changes are characteristic for the adaptive integrated stress response (ISR), which is known to be triggered by unfolded proteins, nutrient and metabolic deficiency, and mitochondrial dysfunctions. However, after a prolonged incubation with myxothiazol (13-17 h), levels of ATF4 mRNA and ATF4-regulated transcripts were found substantially suppressed. The suppression was dependent on the p53 response, which is triggered by the impairment of the complex III-dependent de novo biosynthesis of pyrimidines by mitochondrial dihydroorotate dehydrogenase. The initial adaptive induction of ATF4/ISR acted to promote viability of cells by attenuating apoptosis. In contrast, the induction of p53 upon a sustained inhibition of ETC complex III produced a pro-apoptotic effect, which was additionally stimulated by the p53-mediated abrogation of the pro-survival activities of the ISR. Interestingly, a sustained inhibition of ETC complex I by piericidine did not induce the p53 response and stably maintained the pro-survival activation of ATF4/ISR. We conclude that a downregulation of mitochondrial ETC generally induces adaptive pro-survival responses, which are specifically abrogated by the suicidal p53 response triggered by the genetic risks of the pyrimidine nucleotide deficiency.

Show MeSH
Related in: MedlinePlus