Up-regulation of TNF-α also generates ROS release. Thus the balance in the liver between cell death and survival, with the latter including proliferation and regeneration, determines cell responses. Besides these effects, TNF-α induces hepatocyte proliferation through JNK/SAPK activation and survival pathways through NFκB transcription factor can occur. Permeabilization or rupture of the mitochondrial membrane can occur and provoke liver cell necrosis or apoptosis. Cytokine overproduction can lead to hepatopathies and cancers in which mitochondrial dysfunction is a major mechanism –. TNF-α is particularly involved in cirrhosis. The major targets of TNF-α are neutrophils, endothelial cells, fibroblasts and hepatocytes. In the chronic liver inflammation, the release of pro-inflammatory cytokines such as TNF-α is mainly increased from activated macrophages or monocytes. We suggest that p53 binding to GSK3β, TFAM and D-loop could induce recovery of mtDNA content through mtDNA repair. This study is the first to report the involvement of GSK3β in TNF-α induced mtDNA depletion. The knockdown of p53 by siRNAs, inhibition by the phosphoSer 15p53 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. In addition, mitochondrial D-loop immunoprecipitation (mtDIP) revealed that TNF-α induced p53 binding to the regulatory D-loop region of mtDNA. 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). A transient 8-oxo-dG level of 1.4☐.3 ng/mg DNA and repair of abasic sites were also measured by ELISA assays. Luminol-amplified chemiluminescence (LAC) was used to measure the rapid (10 min) and transient TNF-α induced increase in ROS production (168☑5%). Both lesions and depletion returned to baseline from 1 to 6 h after TNF-α exposure. Using quantitative polymerase chain reaction (qPCR) we found that at 30 min in human hepatoma HepG2 cells TNF-α induced 0.55☐.10 mtDNA lesions per 10 Kb and a 52.4☒.8% decrease in mtDNA content dependent on TNF-R1 receptor and ROS production.
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.
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. 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.