Mitochondrial DNA maintenance is regulated in human hepatoma cells by glycogen synthase kinase 3β and p53 in response to tumor necrosis factor α.
Mitochondrial DNA maintenance is regulated in human hepatoma cells by glycogen synthase kinase 3β and p53 in response to tumor necrosis factor α.
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DOI：
10.1371/journal.pone.0040879
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Reyl-Desmars F
中科院分区：
文献类型：
--
作者：
Vadrot N;Ghanem S;Braut F;Gavrilescu L;Pilard N;Mansouri A;Moreau R;Reyl-Desmars F
在慢性肝脏炎症期间，上调的肿瘤坏死因子α (TNF-α)靶向肝细胞并诱导异常活性氧（ROS）产生，导致线粒体DNA （mtDNA）改变。丝氨酸/苏氨酸糖原合成酶激酶3β (GSK3β)在炎症过程中起关键作用，但其参与mtDNA的维持尚不清楚。本研究的目的是研究其参与TNF-α诱导的mtDNA消耗及其与p53的相互关系，p53是一种已知的维持mtDNA拷贝数的蛋白质。通过定量聚合酶链反应（qPCR），我们发现在人肝癌HepG2细胞中，TNF-α在30min时每10kb诱导0.55±0.10个mtDNA损伤，mtDNA含量减少52.4±2.8%，这取决于TNF- r1受体和ROS的产生。TNF-α暴露后1至6小时，病变和消耗均恢复到基线水平。采用鲁米诺扩增化学发光法（LAC）检测快速（10 min）和瞬时TNF-α诱导的ROS生成增加（168±15%）。酶联免疫吸附法测定瞬时8-o - dg浓度（1.4±0.3 ng/mg）和碱基位点的修复情况。Western Blot观察p53向线粒体的易位，免疫共沉淀法显示TNF-α诱导p53与GSK3β和线粒体转录因子A （TFAM）结合。此外，线粒体d环免疫沉淀（mtDIP）显示TNF-α诱导p53结合mtDNA的调节d环区域。用sirna敲低p53、phosphoSer15p53抗体抑制或转染人突变体活性GSK3β s9a pcDNA3质粒抑制mtDNA含量的恢复，而用SB216763抑制剂阻断GSK3β活性或敲低sirna抑制mtDNA的消耗。这项研究首次报道了GSK3β参与TNF-α诱导的mtDNA缺失。我们认为p53与GSK3β、TFAM和D-loop结合可以通过mtDNA修复诱导mtDNA含量的恢复。
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.
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