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. 2024 Feb 25;56(2):162-173.
doi: 10.3724/abbs.2024012.

AMPK/PGC-1α and p53 modulate VDAC1 expression mediated by reduced ATP level and metabolic oxidative stress in neuronal cells

Zhitong Wang  1   2 , Tingting Xu  1 , Yingni Sun  1 , Xiang Zhang  1 , Xiaoliang Wang  1
Affiliations

AMPK/PGC-1α and p53 modulate VDAC1 expression mediated by reduced ATP level and metabolic oxidative stress in neuronal cells

Zhitong Wang et al. Acta Biochim Biophys Sin (Shanghai). .

Abstract

Voltage-dependent anion channel 1 (VDAC1) is a pore protein located in the outer mitochondrial membrane. Its channel gating mediates mitochondrial respiration and cell metabolism, and it has been identified as a critical modulator of mitochondria-mediated apoptosis. In many diseases characterized by mitochondrial dysfunction, such as cancer and neurodegenerative diseases, VDAC1 is considered a promising potential therapeutic target. However, there is limited research on the regulatory factors involved in VDAC1 protein expression in both normal and pathological states. In this study, we find that VDAC1 protein expression is up-regulated in various neuronal cell lines in response to intracellular metabolic and oxidative stress. We further demonstrate that VDAC1 expression is modulated by intracellular ATP level. Through the use of pharmacological agonists and inhibitors and small interfering RNA (siRNA), we reveal that the AMPK/PGC-1α signaling pathway is involved in regulating VDAC1 expression. Additionally, based on bioinformatics predictions and biochemical verification, we identify p53 as a potential transcription factor that regulates VDAC1 promoter activity during metabolic oxidative stress. Our findings suggest that VDAC1 expression is regulated by the AMPK/PGC-1α and p53 pathways, which contributes to the maintenance of stress adaptation and apoptotic homeostasis in neuronal cells.

Keywords: VDAC1; expression regulation; metabolic oxidative stress; mitochondria.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

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Figure 1
Metabolic oxidative stress reduces intracellular ATP level and upregulates VDAC1 expression (A,B) Cellular ATP levels (A) and western blot analysis of VDAC1 (B) in SK-N-SH cells after 24 h of incubation with antimycin A (15, 30, or 60 μM) or oligomycin (2, 4 or 8 μM) (n=4). The data were analyzed by one-way ANOVA with Bonferroni’s post hoc test and are presented as the mean±SD. (C,D) Cellular ATP levels (C) and western blot analysis of VDAC1 (D) in SK-N-SH cells after 24 h of incubation with 2-DG (4 mM) or culture in DMEM without glucose (glucose deprivation, GD) (n=4). The data were analyzed by t test and are presented as the mean±SD. (E,F) Reduced cellular ATP levels (E) and increased VDAC1 expression (F) were observed in cultured primary rat cortical neurons following treatment with antimycin (25 nM) or oligomycin (10 nM) for 24 h (n=4). (G,H) After incubation with antimycin (60 μM), oligomycin (8 μM), or 2-deoxyglucose (4 mM) for 24 h, mRNA expression of VDAC1 in SK-N-SH cells was evaluated by real-time PCR (n=5) and cell viability was evaluated by MTT assay (n=4). (I) SK-N-SH cells were treated with cisplatin (20 μM, as a positive control), oligomycin (8 μM), antimycin A (60 μM), or 2-DG (4 mM) for 24 h and then incubated with Fluo-4 and analyzed by flow cytometry (n=4). The data were analyzed by one-way ANOVA with Bonferroni’s post hoc test and are presented as the mean±SD. *P<0.05; **P<0.01.
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Figure 2
DCA and glucose increase intracellular ATP level and downregulate VDAC1 expression (A,B) Cellular ATP levels (A) and western blot analysis of VDAC1 (B) in SK-N-SH cells after 24 h of incubation with DCA (1.25, 2.5, or 5 mM) (n=4). The data were analyzed by one-way ANOVA with Bonferroni’s post hoc test and are presented as the mean±SD. (C,D) Cellular ATP levels (C) and VDAC1 expression (D) were obtained in cultured primary rat cortical neurons following treatment with DCA (100 μM) for 24 h (n=4). The data were analyzed by t test and are presented as the mean±SD. (E,F) Changes in ATP levels (E) and VDAC1 expression (F) in SH-SY5Y cells cultured for 72 h under normal (7.5 mM) or high glucose (20 mM or 50 mM) conditions (n=4). The data were analyzed by one-way ANOVA with Bonferroni’s post hoc test and are presented as the mean±SD. (G,H) SK-N-SH cells were incubated for 24 h with DCA (5 mM), mRNA expression of VDAC1 was evaluated by real-time PCR (n=5), and cell viability was evaluated by MTT assay (n=4). (I) To analyze changes in cytosolic Ca2+, SK-N-SH cells were treated for 24 h with DCA (5 mM) followed by incubation with Fluo-4 (n=4). The data were analyzed by t tests and are presented as the mean±SD. *P<0.05; **P<0.01.
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Figure 3
Changes in VDAC1 expression modulates intracellular metabolism or the mitochondrial membrane potential (A) Intracellular ATP levels in SK-N-SH cells were measured after 24 h of transfection with VDAC1 siRNA (20 or 50 nM). The data were analyzed by t test and are presented as the mean±SD. (B) The expressions of VDAC1, p-ACC, and ACC were examined by western blot analysis after 24 h of transfection with VDAC1 siRNA (n=4). The data were analyzed by one-way ANOVA with Bonferroni’s post hoc test and are presented as the mean±SD. (C) The mitochondrial membrane potential was determined in SK-N-SH cells incubated with VDAC1 siRNA (50 nM) for 24 h. (D) Intracellular ATP levels in SK-N-SH cells were tested after 24 h of transfection with pcDNA3.1-VDAC1-3×Flag plasmids (2 μg) or vector for 48 h. (E) VDAC1, p-ACC, and ACC expressions were determined via western blot analysis after 48 h of transfection with VDAC1-3×Flag plasmids (*P<0.05 vs vector, n=4). (F) Mitochondrial membrane potential was examined in SK-N-SH cells incubated with VDAC1-3×Flag plasmids (2 μg) or vector for 48 h (n=4). The data were analyzed by t test and are presented as the mean±SD. *P<0.05; **P<0.01.
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Figure 4
Intracellular ATP level modulates the AMPK/PGC-1α signaling pathway, influencing VDAC1 expression (A) Western blot analysis of VDAC1 in SK-N-SH cells incubated with AICAR (2 mM) or compound C (20 μM) for 24 h. The data were analyzed by t test and are presented as the mean±SD. (B) Western blot analysis showed that infection with PGC-1α small interfering RNA (20 or 50 nM) for 24 h decreased PGC-1α and VDAC1 expressions (n=4). The data were analyzed by one-way ANOVA with Bonferroni’s post hoc test and are presented as the mean±SD. *P<0.05; **P<0.01.
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Figure 5
p53 is a potential VDAC1 transcription factor (A) Potential transcription factors associated with VDAC1 were identified via the PROMO prediction web tool (http://alggen.lsi.upc.es). (B,C) The JASPAR prediction web tool (http://jaspar.genereg.net) showed that the metabolism-related transcription factor TP53 gained high scores for binding to the VDAC1 gene promoter. (D) Data from the Genotype-Tissue Expression (GTEx) project database showed a positive correlation between TP53 and VDAC1 expression in various brain regions, including the cortex, hippocampus, caudate (basal ganglia), amygdala, nucleus accumbens (basal ganglia), hypothalamus, and substantia nigra.
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Figure 6
The expression of p53 is regulated by metabolic regulators (A) Fluorescence images were obtained for different metabolic regulators. SK-N-SH cells were treated with antimycin (60 μM) or DCA (5 mM) for 24 h. The cells were fixed and labeled with anti-p53 and anti-VDAC1 antibodies, stained with Hoechst 33258 DNA stain, and visualized via confocal microscopy. Scale bar: 10 μm. (B) Changes in p53 expression in SK-N-SH cells incubated with antimycin (60 μM) or DCA (5 mM) for 24 h determined by immunoblotting (n=4). The data were analyzed by t test and are presented as the mean±SD. *P<0.05; **P<0.01.
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Figure 7
VDAC1 expression is regulated by p53 (A) p53, VDAC1 and Bax expressions in SK-N-SH cells transfected with pcDNA3.1-p53-3×Flag plasmids (2 μg) or vector for 48 h (n=4). The data were analyzed by t test and are presented as the mean±SD. *P<0.05; **P<0.01. (B) Western blot analysis showed that SK-N-SH cells infected with p53 siRNA (20 or 50 nM) for 24 h had decreased p53, VDAC1, and Bax expressions (n=4). The data were analyzed by one-way ANOVA with Bonferroni’s post hoc test and are presented as the mean±SD. *P<0.05. (C) SK-N-SH cells were transfected with pGL3-basic-VDAC1-Luc (500 ng) or pRL-SV40 (50 ng, as the control for transfection efficiency), along with the cDNA constructs (p53, 500 ng; Vector, 500 ng). The cells were lysed, and luciferase activity was determined 24 h after transfection (n=4). The data were analyzed by t test and are presented as the mean±SD. **P<0.01. (D) ChIP analysis of SK-N-SH cells showing the physical binding of the p53 transcription factor to the VDAC1 promoter; IgG was used as the negative control. (E) The effect of oligomycin (2 or 4 μM) and 2-DG (4 mM) on VDAC1 expression is mediated through p53. After 24 h of transfection with NC or p53 siRNA (50 nM), SK-N-SH cells were incubated with oligomycin (2 or 4 μM) or 2-DG (4 mM) for 24 h. VDAC1 expression was examined via western blot analysis (n=4). The data were analyzed by one-way ANOVA with Bonferroni’s post hoc test and are presented as the mean±SD. *P<0.05 vs the negative control group; $$P<0.01 vs the NC+oligomycin group; ##P<0.01 vs the NC+2-DG group.
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Figure 8
The AMPK/PGC-1α and p53 pathways modulate VDAC1 expression in neuronal cells under metabolic oxidative stress AMPK is a critical cellular energy sensor whose phosphorylation, activation or inhibition are regulated by the agonist AICAR or the inhibitor compound C. AMPK has been proven to be activated by metabolic stress. As a transcriptional coactivator of AMPK, PGC-1α participates in mitochondrial biogenesis and is induced by AMPK activation. Metabolic oxidative stress increases the tumor suppressor p53 expression. In this study, we found that the AMPK/PGC-1α and p53 pathways mediate the upregulation of VDAC1, known as the mitochondrial gatekeeper, in metabolic oxidative stress. Overexpression of VDAC1 caused the release of apoptotic proteins, such as cytochrome c, and induced cell apoptosis. Conversely, inhibition of AMPK or increased oxidative phosphorylation (OXPHOS) downregulates VDAC1 expression to reduce the mitochondrial permeability of ATP and ROS.
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This work was supported by the grant from the National Natural Science Foundation of China (No. 81573417).
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