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

作者：Vadrot Nathalie^*; Ghanem Sarita; Braut Francoise; Gavrilescu Laura; Pilard Nathalie; Mansouri Abdellah; Moreau Richard; Reyl Desmars Florence

来源：PLos One, 2012, 7(7): e40879.

DOI：10.1371/journal.pone.0040879

During chronic liver inflammation, up-regulated Tumor Necrosis Factor alpha (TNF-alpha) targets hepatocytes and induces abnormal reactive oxygen species (ROS) production responsible for mitochondrial DNA (mtDNA) alterations. The serine/threonine Glycogen Synthase Kinase 3 beta (GSK3 beta) 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-alpha 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-alpha 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-alpha exposure. Luminol-amplified chemiluminescence (LAC) was used to measure the rapid (10 min) and transient TNF-alpha 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-alpha induced p53 binding to GSK3 beta and mitochondrial transcription factor A (TFAM). In addition, mitochondrial D-loop immunoprecipitation (mtDIP) revealed that TNF-alpha 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 GSK3 beta S9A pcDNA3 plasmid inhibited recovery of mtDNA content while blockade of GSK3 beta activity by SB216763 inhibitor or knockdown by siRNAs suppressed mtDNA depletion. This study is the first to report the involvement of GSK3 beta in TNF-alpha induced mtDNA depletion. We suggest that p53 binding to GSK3 beta, TFAM and D-loop could induce recovery of mtDNA content through mtDNA repair.

• 出版日期2012-7-20