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.
INSERM U773, CRB3, Equipe Moreau, Université Paris 7 Denis Diderot, Faculté de Médecine X Bichat, Paris, France.
Nathalie
N
Vadrot
Sarita
S
Ghanem
Françoise
F
Braut
Laura
L
Gavrilescu
Nathalie
N
Pilard
Abdellah
A
Mansouri
Richard
R
Moreau
Florence
F
Reyl-Desmars
D000080242:8-Hydroxy-2'-Deoxyguanosine; D017209:Apoptosis / Q000187:drug effects; D006528:Carcinoma, Hepatocellular / Q000235:genetics* / Q000378:metabolism*; D004249:DNA Damage / Q000187:drug effects; D004260:DNA Repair / Q000187:drug effects; D004272:DNA, Mitochondrial / Q000187:drug effects / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D003849:Deoxyguanosine / Q000031:analogs & derivatives / Q000378:metabolism; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism*; D000071679:Glycogen Synthase Kinase 3 beta; D056945:Hep G2 Cells; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics* / Q000378:metabolism*; D024101:Mitochondrial Proteins / Q000378:metabolism; D011485:Protein Binding; D021381:Protein Transport; D034622:RNA Interference; D017382:Reactive Oxygen Species / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*
D004272:DNA, Mitochondrial; D004268:DNA-Binding Proteins; D024101:Mitochondrial Proteins; D017382:Reactive Oxygen Species; C499631:TFAM protein, human; D014157:Transcription Factors; D014409:Tumor Necrosis Factor-alpha; D016159:Tumor Suppressor Protein p53; D000080242:8-Hydroxy-2'-Deoxyguanosine; C000605506:GSK3B protein, human; D000071679:Glycogen Synthase Kinase 3 beta; D038362:Glycogen Synthase Kinase 3; D003849:Deoxyguanosine