Mitochondrial DNA Maintenance Is Regulated in Human Hepatoma Cells by Glycogen Synthase Kinase 3β and p53 in Response to Tumor Necrosis Factor α

Nathalie Vadrot,Sarita Ghanem,Nathalie Pilard,Françoise Braut,Abdellah Mansouri,Richard Moreau,Laura Gavrilescu,Florence Reyl-Desmars,Janine Santos

doi:10.1371/journal.pone.0040879

Nathalie Vadrot, Sarita Ghanem + Show 7 more

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https://doi.org/10.1371/journal.pone.0040879

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Journal: PloS one	Publication Date: Jul 20, 2012
Citations: 41	License type: CC BY 4.0

Affiliation: Délégation Paris 7, Inserm, Université Paris Cité

Abstract

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 phosphoSer15p53 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.

Highlights

In the chronic liver inflammation, the release of pro-inflammatory cytokines such as Tumor Necrosis Factor alpha (TNF-a) is mainly increased from activated macrophages or monocytes [1]
The lack of apoptotic bodies observed under UV-microscopy after 30 or 100 ng/ml of TNF-a treatment and DAPI staining is consistent with an absence of apoptosis after 18-h of TNF-a treatment (Figure 1C)
The aim of this study was to investigate whether Glycogen Synthase Kinase 3 beta (GSK3b) is involved in the loss of TNF-a induced mitochondrial DNA (mtDNA) content and counteracted by p53

Summary

Introduction

In the chronic liver inflammation, the release of pro-inflammatory cytokines such as TNF-a is mainly increased from activated macrophages or monocytes [1]. The major targets of TNF-a are neutrophils, endothelial cells, fibroblasts and hepatocytes [1,2]. Permeabilization or rupture of the mitochondrial membrane can occur and provoke liver cell necrosis or apoptosis [1,2,7]. Besides these effects, TNF-a induces hepatocyte proliferation through JNK/ SAPK activation and survival pathways through NFkB transcription factor can occur [1,2]. The balance in the liver between cell death and survival, with the latter including proliferation and regeneration, determines cell responses [1,2]

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