Metallothionein isoform 2A expression is inducible and protects against ROS-mediated cell death in rotenone-treated HeLa cells.
The role of MT (metallothionein) gene expression was investigated in rotenone-treated HeLa cells to induce a deficiency of NADH:ubiquinone oxidoreductase (complex I). Complex I deficiency leads to a diversity of cellular consequences, including production of ROS (reactive oxygen species) and apoptosis. HeLa cells were titrated with rotenone, resulting in dose-dependent decrease in complex I activity and elevated ROS production at activities lower than 33%. Expression of MT2A (MT isoform 2A), but not MT1A or MT1B RNA, was significantly inducible by rotenone (up to 7-fold), t-BHP (t-butyl hydroperoxide; 5-fold) and CdCl2 (50-fold), but not ZnCl2. Myxothiazol treatment did not elevate either ROS or MT2A levels, which supports a ROS-related mechanism for rotenone-induced MT2A expression. To evaluate the role of MT2A expression, MT2A and MT1B were overexpressed in HeLa cells and treated with rotenone. Compared with control and MT1B-overexpressing cells, ROS production was significantly lower and cell viability higher in MT2A-overexpressing HeLa cells when ROS production was enhanced by treatment with t-BHP. Mitochondrial membrane potential was noticeably less reduced in both MT-overexpressing cell lines. MT2A overexpression in rotenone-treated cells also significantly reduced or delayed apoptosis induction, as measured by caspase 3/7 activity and cytosolic nucleosome enrichment. We conclude that MT2A offers significant protection against the main death-causing consequences of rotenone-induced complex I deficiency in HeLa cells. Our results are in support of the protective role against oxidative stress ascribed to MTs and provide evidence that MT2A expression may be a beneficial downstream adaptive response in complex I-deficient cells.
Reinecke
Fimmie
F
Division of Biochemistry, School for Chemistry and Biochemistry, North-West University, Potchefstroom 2531, South Africa.
Levanets
Oksana
O
Olivier
Yolanda
Y
Louw
Roan
R
Semete
Boitumelo
B
Grobler
Anne
A
Hidalgo
Juan
J
Smeitink
Jan
J
Olckers
Antonel
A
Van der Westhuizen
Francois H
FH
D000255:Adenosine Triphosphate / Q000032:analysis; D017209:Apoptosis / Q000187:drug effects; D016923:Cell Death / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D042967:Electron Transport Complex I / Q000187:drug effects; D014450:Electron Transport Complex III / Q000187:drug effects; D006367:HeLa Cells; D006801:Humans; D008564:Membrane Potentials / Q000187:drug effects; D008668:Metallothionein / Q000235:genetics / Q000378:metabolism*; D008928:Mitochondria / Q000378:metabolism; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012402:Rotenone / Q000494:pharmacology*; D015854:Up-Regulation / Q000187:drug effects*; D020122:tert-Butylhydroperoxide / Q000494:pharmacology
D020033:Protein Isoforms; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D011994:Recombinant Proteins; D012402:Rotenone; D000255:Adenosine Triphosphate; D008668:Metallothionein; D020122:tert-Butylhydroperoxide; D042967:Electron Transport Complex I; D014450:Electron Transport Complex III