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. 2012 Dec 11;109(50):20491-6.
doi: 10.1073/pnas.1206530109. Epub 2012 Nov 26.

Mitochondrial localization of TIGAR under hypoxia stimulates HK2 and lowers ROS and cell death

Eric C Cheung  1 , Robert L Ludwig, Karen H Vousden
Affiliations

Mitochondrial localization of TIGAR under hypoxia stimulates HK2 and lowers ROS and cell death

Eric C Cheung et al. Proc Natl Acad Sci U S A. .

Abstract

The p53-inducible protein TIGAR (Tp53-induced Glycolysis and Apoptosis Regulator) functions as a fructose-2,6-bisphosphatase (Fru-2,6-BPase), and through promotion of the pentose phosphate pathway, increases NADPH production to help limit reactive oxygen species (ROS). Here, we show that under hypoxia, a fraction of TIGAR protein relocalized to mitochondria and formed a complex with hexokinase 2 (HK2), resulting in an increase in HK2 activity. Mitochondrial localization of TIGAR depended on mitochondrial HK2 and hypoxia-inducible factor 1 (HIF1α) activity. The ability of TIGAR to function as a Fru-2,6-BPase was independent of HK2 binding and mitochondrial localization, although both of these activities can contribute to the full activity of TIGAR in limiting mitochondrial ROS levels and protecting from cell death.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Relocalization of TIGAR to mitochondria during hypoxia. (A) Western blot analysis on whole cell lysate (Left) and on mitochondrial fraction (Right) of A2780 and SW48 cells after 24 h in control (normoxia) and 0.1% O2. mtHsp70 was used as a loading control for mitochondria. SW48 (B) and A2780 (C) cells were incubated in control and 0.1% O2. After 24 h, cells were fixed and stained with anti-TIGAR antibody and anti-Tom20 antibody to visualize mitochondria. (D) HeLa cells were treated with 100 μM DPCA. After 24 h, cells were fixed and stained with anti-TIGAR and anti-Tom20 antibody to visualize mitochondria. DMSO only was used as control. (E) A2780 cells were treated with 1 mM DMOG. After 24 h, cells were fixed and stained with anti-TIGAR and anti-Tom20 antibody to visualize mitochondria. (F) HeLa cells exposed to 1% O2 for 24 h were fixed and then permeabilized by using the indicated concentration of digitonin. Cells were then stained with the indicated antibody to visualize different mitochondrial compartments. (Scale bars: B and D, 40 μm; C, 10 μm; E, 20 μm.)
Fig. 2.
Fig. 2.
TIGAR interacts with HK2 during hypoxia. (A) Cell lysates from A2780, CaCO2, and SW48 cells with indicated treatments (hypoxia = 0.1% O2) were immunoprecipitated with anti-TIGAR antibody. The membranes were probed with the indicated antibodies. (B) HeLa cells grown in the indicated conditions (hypoxia = 0.1% O2) for 24 h were fixed and stained with the indicated antibodies. (C) HeLa cells grown in the indicated conditions (hypoxia = 0.1% O2) for 24 h were fixed and stained with anti-HK2 and anti-mtHsp70 to visualize mitochondria. (D) HeLa cells treated with control siRNA or HK2 siRNA that were grown in normoxia or 0.1% O2 for 24 h were fixed and stained with anti-TIGAR antibody and anti-Tom20 antibody to visualize mitochondria. (E) Cells transfected with the indicated plasmids or siRNA were grown in normoxia or hypoxia (0.1% O2) for 24 h and hexokinase (HK) activity measured. The relative activity was obtained by normalization to the cells with control siRNA. *P < 0.05 compared with control. (Scale bars: 20 μm.)
Fig. 3.
Fig. 3.
TIGAR interaction with HK2 during hypoxia depends on HIF1α. (A) A2780 cells treated with the indicated compounds that were grown in 0.1% O2 for 24 h were fixed and stained with anti-Tom20 to visualize mitochondria and anti-TIGAR to determine TIGAR location. (B) A2780 cells treated with either control siRNA or HIF1α siRNA were grown in hypoxia (0.1% O2) for 24 h, then fixed and stained with anti-TIGAR and anti-Tom20 to visualize mitochondria. (C) Cell lysates from A2780 cells treated with control siRNA or HIF1α siRNA and grown in normoxic or hypoxic conditions were immunoprecipitated with anti-TIGAR antibody. The membranes were probed with the indicated antibodies. (D) Cell lysates from A2780 cells treated with CAY10582 as indicated and grown in normoxic or hypoxic (0.1% O2) conditions were immunoprecipitated with anti-TIGAR antibody. The membranes were probed with the indicated antibodies. (Scale bars: 15 μm.)
Fig. 4.
Fig. 4.
TIGAR maintains cell survival via HK2 during hypoxia. (A) Cells transfected with empty vector and control siRNA (control), wild-type TIGAR, HK2, or indicted siRNAs were grown in normoxia (open bar) or hypoxia (0.1% O2) (filled bars) for 24 h. (A) Mitochondrial ROS level. (B) Mitochondrial membrane potential was measured by TMRE fluorescence. (C) Cell death was measured by PI exclusion. *P < 0.05 compared with hypoxia control. (D) Western blot showing representative example of expression of TIGAR and HK2 protein in cells treated in A–C.
Fig. 5.
Fig. 5.
Relocalization of TIGAR mutants during hypoxia. (A) Schematic of the various TIGAR mutants. (B) HeLa cells (Left) and A2780 cells (Right) transfected with wild-type TIGAR or the indicated mutants were grown in hypoxia (1% O2) at 30 °C for 24 h. Cells were then fixed and stained with anti-TIGAR and anti-Tom20 to visualize mitochondria. (Scale bar: 15 μm.) (C) Cell lysates from HeLa cells transfected with wild-type TIGAR or the indicated mutants and grown at 30 °C in hypoxia (1% O2) were immunoprecipitated by using anti-TIGAR antibody. The membranes were probed with the indicated antibodies. (D) Cells transfected with wild-type TIGAR or the indicated mutants were grown in normoxia (control) or hypoxia (1% O2) for 24 h at 30 °C and their HK activity measured. The relative activity was obtained by normalization to the control cells in normoxia. *P < 0.05 compared with hypoxia control. (E) Fru-2,6-BP level in cells transfected with the indicated TIGAR constructs (empty vector as control) that were exposed to hypoxia (1% O2) for 24 h. *P < 0.05 compared with control.
Fig. 6.
Fig. 6.
TIGAR mutant that is unable to bind to HK2 fails to protect cells during hypoxia. Cells transfected with the indicted TIGAR mutants were grown in control or hypoxia (1% O2) for 24 h at 30 °C. (A) Mitochondrial ROS level. (B) Mitochondrial membrane potential was measured by TMRE fluorescence. (C) Cell death was measured by PI exclusion. *P < 0.05 compared with hypoxia control. (D) Western blot showing representative example of expression of TIGAR and HK2 protein in cells treated in A–C.
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