Mitochondrial DNA maintenance is regulated in human hepatoma cells by glycogen synthase kinase 3β and p53 in response to tumor necrosis factor α.

During chronic liver inflammation, up-regulated Tumor Necrosis Factor alpha (TNF-α) targets hepatocytes and induces abnormal reactive oxygen species (ROS) production responsible for mitochondrial DNA (mtDNA) alterations. The serine/threonine Glycogen Synthase Kinase 3 beta (GSK3β) plays a pivotal role during inflammation but its involvement in the maintenance of mtDNA remains unknown. The aim of this study was to investigate its involvement in TNF-α induced mtDNA depletion and its interrelationship with p53 a protein known to maintain mtDNA copy numbers. Using quantitative polymerase chain reaction (qPCR) we found that at 30 min in human hepatoma HepG2 cells TNF-α induced 0.55±0.10 mtDNA lesions per 10 Kb and a 52.4±2.8% decrease in mtDNA content dependent on TNF-R1 receptor and ROS production. Both lesions and depletion returned to baseline from 1 to 6 h after TNF-α exposure. Luminol-amplified chemiluminescence (LAC) was used to measure the rapid (10 min) and transient TNF-α induced increase in ROS production (168±15%). A transient 8-oxo-dG level of 1.4±0.3 ng/mg DNA and repair of abasic sites were also measured by ELISA assays. Translocation of p53 to mitochondria was observed by Western Blot and co-immunoprecipitations showed that TNF-α induced p53 binding to GSK3β and mitochondrial transcription factor A (TFAM). In addition, mitochondrial D-loop immunoprecipitation (mtDIP) revealed that TNF-α induced p53 binding to the regulatory D-loop region of mtDNA. The knockdown of p53 by siRNAs, inhibition by the phosphoSer(15)p53 antibody or transfection of human mutant active GSK3βS9A pcDNA3 plasmid inhibited recovery of mtDNA content while blockade of GSK3β activity by SB216763 inhibitor or knockdown by siRNAs suppressed mtDNA depletion. This study is the first to report the involvement of GSK3β in TNF-α induced mtDNA depletion. We suggest that p53 binding to GSK3β, TFAM and D-loop could induce recovery of mtDNA content through mtDNA repair

TNF-α induced ROS, 8-oxo-dG production and mtDNA repair.

<p>(A) TNF-α induced extra and intracellular ROS were measured over 1 h-period using LAC assay on cell suspension (10⁶ cells in 0.5 ml Hanks buffer) as described in Materials and Methods. One representative experiment of four independent studies is shown. (B) Chemiluminescence is also quantified at the peak in the absence or presence of 5 mM NAC as a percentage of basal value (control) (mean ± SEM for three independent experiments, *p<0.05). (C) TNF-α induced levels of 8-oxo-dG after 15 min to 3 h cell treatment were measured using the OxiSelect™ Oxidative DNA damage ELISA kit (mean values ± SEM of three independent experiments *p<0.05). (D) The decrease of mtDNA remaining ARP-reactive sites created at 30 min TNF-α treatment (100%) was measured from 1 to 6 h using the OxiSelect™ oxidative DNA damage quantitation kit (mean values ± SEM of three independent experiments *p<0.05 <i>vs</i> 30 min).</p

p53 inhibition by siRNAs and phosphoSer¹⁵p53 antibody prevented the reversion of mtDNA depletion.

<p>(A) All siRNA transfections were performed with 12.5 nM siRNAs directed against p53 mRNA (p53 siRNAs) or non-targeting siRNAs (NT) and DharmaFECT4® transfection reagent used as a control (c). To check siRNA transfection efficiency, Western Blots were performed at 48 h using p53 or GAPDH (loading control) antibody. (B) The knockdown of p53 expression by siRNAs relative to GAPDH was quantified using the Bio1D software (mean values ± SEM of three experiments *p<0.05) (C) DNA was isolated from untransfected cells or transfected with NT siRNAs or with p53 siRNAs. Cells were also permeabilized with 0.1% Triton X100 and pretreated for 1 h with 1 µg/ml phosphoSer¹⁵p53 antibody (phosphoSer¹⁵Ab). Cells were then treated for 0 to 6 h with 30 ng/ml TNF-α. The quantification of mtDNA content was performed by simultaneous real-time qPCR amplification of fragments encoding mitochondrial 12S rRNA and nuclear 18S rRNA serving as a reference gene. Results are expressed in mtDNA over nDNA relative ratio (mean values ± SEM of three independent experiments with five replicates, *p<0.05).</p

TNF-α induced p53 interaction to GSK3β, TFAM and D-loop.

<p>Cells were treated for 0 (zero-time control) or 1 h with 30 ng/ml TNF-α. Mitochondrial fractions were isolated and co-immunoprecipitated or not (input) with GSK3β, p53 (FL-393) or TFAM polyclonal antibodies, IgG or no antibody was used as a control (c). (A) Western Blots were performed using GSK3β or p53 (DO1) antibody. (B) Western Blots were performed using phosphoSer¹⁵p53 or phosphoSer⁹GSK3β antibody in not pretreated or pretreated cells with GSK3β inhibitor SB216763. (C) Western Blots were performed using TFAM or GSK3β antibody. (D) The mtDIP assay was performed with cross-linked DNA prepared from treated cells for 0 (zero-time control) or 1 h with 30 ng/ml TNF-α. Immunoprecipitates were performed without (control, c) or with IgG used as a control or with p53 antibody. PCR were realized on immunoprecipitates or inputs using a primer pair covering D-loop, cytochrome b (cyt b) or ATPase 6 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040879#pone-0040879-g005" target="_blank">Figure 5D</a>).</p

TNF-α induced p53 translocation to mitochondria.

<p>(A) Accumulation of p53 was investigated by Western Blot using p53 antibody in lysate from cells treated for 1 h with 5–100 ng/ml TNF-α or with 30 ng/ml TNF-α for 0 to 180 min. β-actin served as a loading control (B) Accumulation of phosphoSer¹⁵p53 was investigated by Western Blot in lysate from cells treated for 0–180 min with 30 ng/ml TNF-α. β-actin served as a loading control. (C) Mitochondrial (mito) and cytoplasmic fractions (cyto) were isolated as described in Material and Methods and their purities checked by Western Blot using β-actin antibody (cytoplasm) and COXI (mitochondria). (D) The translocation of p53 to mitochondria was investigated by Western Blot using p53 antibody on mitochondrial (mito) or cytoplasmic (cyto) fractions isolated from cells treated or not for 0 to 60 min with 30 ng/ml TNF-α. COXI and β-actin served as loading controls. (E) Cells were pretreated or not (control) for 1 h with 5 mM NAC before exposure to TNF-α. Western Blots were performed using p53 antibody. COXI was used as a loading control.</p

SB216763 or GSK3β siRNAs inhibited mtDNA depletion whereas GSK3βS9A suppressed the reversion.

<p>(A) All siRNA transfections were performed or not (control, C) with 12.5 nM siRNAs directed against GSK3β mRNA (GSK3β siRNAs) or non-targeting (NT) siRNAs in DharmaFECT4® transfection reagent. To check SiRNA transfection efficiency, Western Blots were performed at 48 h with GSK3β or GAPDH (loading control) antibody. (B) Inhibition of GSK3β expression by siRNAs relative to GAPDH was quantified using the Bio1D software (mean values ± SEM of three experiments *p<0.05) (C) Cells were transfected or not (control C or Fugene HD® alone, F) with the mutant GSK3βS9A pcDNA3 plasmid or the empty plasmid (pcDNA3) using Fugene HD® (F). After 72 h transfection, the expression of recombinant GSK3βS9A protein was checked by Western Blot using GSK3β or GAPDH (loading control) antibody. (D) Cells were treated or not with SB216763 or transfected or not (untransfected cells) with NT siRNAs or GSK3β siRNAs or GSK3βS9A pcDNA3 plasmid. Then, they were treated for 0 (zero-time control) to 6 h with 30 ng/ml TNF-α. Total DNA was isolated and the quantification of mtDNA content performed by real-time qPCR co-amplification of fragments encoding mitochondrial 12S rRNA and nuclear 18S rRNA as a gene reference. Results are expressed in mtDNA over nDNA relative ratio (mean values ± SEM of three independent experiments with five replicates, *p<0.05).</p

TNF-α induced mtDNA depletion, lesions and repair.

<p>(A) Cells were pretreated or not (TNF-α) for 1 h with 1 µg/ml TNF-R1 antibody (TNF-R1 Ab) or with 5 mM NAC. They were then treated for 0 to 6 h with 30 ng/ml TNF-α. To evaluate mtDNA depletion, total genomic DNA was isolated and quantification of mtDNA performed by simultaneous real-time qPCR amplification of fragments encoding mitochondrial 12S rRNA and nuclear 18S rRNA used as a reference gene. Results are expressed in 12S mtDNA over 18S nDNA relative ratio (mean values ± SEM of four independent experiments with four replicates, *p<0.05). (B) mtDNA lesions per 10 Kb were quantified by qPCR amplification of a large fragment (8.9 Kb) from cells treated or not for 15 min-6 h with 30 ng/ml TNF-α and expressed using the Poisson expression <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040879#pone.0040879-Santos1" target="_blank">[27]</a> (mean values ± SEM of three independent experiments with three replicates, *p<0.05). (C) mtDNA repair activity was measured by calculating relative amplification comparing the values of the treated samples with undamaged control <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040879#pone.0040879-Santos1" target="_blank">[27]</a>, a 50% mtDNA control has been performed (mean values ± SEM of three independent experiments with three replicates, *p<0.05).</p

TNF-α did not induce apoptosis of HepG2 cells.

<p>(A) Western Blot using the TNF-R1 receptor antibody was performed on cell lysate. (B) PARP cleavage was investigated by Western Blot in cells treated for 18 h with 30 or 100 ng/ml TNF-α or with 1 µM doxorubicin (Doxo) as a positive control. (C) The lack of apoptotic bodies in basal or cells treated with 30 or 100 ng/ml TNF-α has been confirmed by DAPI staining and UV-microscopy.</p