p53-dependent regulation of mitochondrial energy production by the RelA subunit of NF-κB

Abstract

Aberrant activity of the nuclear factor kappaB (NF-κB) transcription factor family, which regulates cellular responses to stress and infection, is associated with many human cancers. In this study, we define a function of NF-κB in regulation of cellular respiration that is dependent upon the tumor suppressor p53. Translocation of the NF-κB family member RelA to mitochondria was inhibited by p53 by blocking an essential interaction with the HSP Mortalin. However, in the absence of p53, RelA was transported into the mitochondria and recruited to the mitochondrial genome where it repressed mitochondrial gene expression, oxygen consumption, and cellular ATP levels. We found mitochondrial RelA function to be dependent on its conserved C-terminal transactivation domain and independent of its sequence-specific DNA-binding ability, suggesting that its function in this setting was mediated by direct interaction with mitochondrial transcription factors. Taken together, our findings uncover a new mechanism through which RelA can regulate mitochondrial function, with important implications for how NF-κB activity and loss of p53 can contribute to changes in tumor cell metabolism and energy production.

Figure 1

RelA regulates cellular oxygen consumption and ATP Production (A) RelA knockdown in U-2 OS cells significantly decreased oxygen consumption (p<0.01, n=8) in early passage cells but increased oxygen consumption (p<0.001, n=8) in late passage cells. The control levels in earlier and later passage cells have both been normalized to 1. Actual values (NRF/cell) for control early passage cells were 0.25 +/− 0.10 and for later passage cells were 0.13 +/− 0.05. (B) RelA knockdown in late passage U-2 OS cells increased oxygen consumption (p<0.001) that was blocked by 4 hour incubation with the H⁺-ATP synthase inhibitor, oligomycin (0.5 μg/ml). (C) RelA knockdown in U-2 OS cells significantly decreased ATP levels (p<0.01, n=9) in early passage cells but increased ATP levels (p<0.01, n=8) in late passage cells. The control levels in earlier and later passage cells have both been normalized to 1. Actual values (RLU/cell) for control early passage cells were 145.91 +/− 37.12 and for later passage cells were 113.61 +/− 26.74. (D) RelA knockdown in late passage U-2 OS cells increased ATP production (p<0.01) that was blocked by 4 hour incubation with the H⁺-ATP synthase inhibitor, oligomycin (0.5 μg/ml).

Figure 2

RelA binds to the mitochondrial genome and regulates mitochondrial gene expression (A) Mitochondrial extracts were isolated from U-2 OS cells at early and late passage number and equivalent levels of protein (4μg) were resolved by SDS-PAGE before western blotting for the indicated proteins. Controls for mitochondria (VDAC), cytoplasm (α-tubulin) and organelle contamination (BiP) were used. (B & C) ChIP analysis of RelA (B, p<0.05, n=8/group) or POLRMT (C, p<0.05, n=4/group) binding to the D-Loop region of the mitochondrial genome in U-2 OS cells. (D) RelA knockdown induces expression of the mitochondrial gene cytochrome B in late passage U-2 OS cells (p<0.01, n=6/group). (E) ChIP analysis of RelA binding to the D-loop region of the mitochondrial genome in U-2 OS cells transfected with 5μg of RSV expression plasmid encoding a mitochondrial targeting sequence (MTS) fused to the N-terminal of RelA, RelA DNA binding mutant (DBM), RelA Rel homology domain (RHD) or control MTS alone (MTS-RSV) (p<0.001, n=7/group). (F) Expression of an MTS-RelA or MTS-RelA DBM plasmid significantly decreased cytochrome B mRNA levels in U-2 OS cells (p<0.01, n=7/group). Data was normalized relative to 18s mRNA and expressed relative to the MTS-RSV control.

Figure 3

RelA mitochondrial localization is p53 dependent. (A & B) Equivalent levels of mitochondrial extracts (8μg) from H1299 cells (A) and p53 −/− MEFs (B) transfected with 5μg of pcDNA3 or pcDNA3-p53 expression plasmid were resolved by SDS-PAGE and western blotted to determine RelA protein levels. (C) ChIP analysis demonstrating significant RelA binding to the cytochrome B region, which was inhibited upon induction of p53, in H1299^wtp53 cells prepared with and without IPTG induction of p53 (p<0.05, n=4/group).

Figure 4

RelA regulation of mitochondrial energy production is p53 dependent. (A & B) The effect of RelA knockdown on cytochrome B mRNA levels was examined in H1299 cells (A; p<0.05, n=6/group) and p53 −/− MEF cells (B; p<0.05, n=5/group) following transfection with 5μg of pcDNA3 or pcDNA3-p53 expression plasmid. Data were normalized relative to 18s mRNA and expressed relative to the pcDNA3 control. (C & D) Oxygen consumption (C; p<0.001, n=4/group) and ATP production (D; p<0.01, n=4/group) were measured after RelA knockdown in H1299^wtp53 cells with and without IPTG induction of p53 (200 μM for 24 hours).

Figure 5

The Heat Shock 70 family member Mortalin is a RelA binding protein (A) The RelA T505 region in the TAD is highly conserved across species. A peptide spanning this region (T505 peptide) and a control scramble peptide (Scramble) were used to identify binding partners of this region using peptide affinity chromatography. (B) A 75 kDa protein from HeLa whole cell extracts that bound to the T505 peptide was identified by mass spectrometry as Mortalin. (C) Western blot of eluted column fractions confirming Mortalin binding to the RelA T505 motif peptide (upper panel) and co-immunoprecipitation of endogenous RelA with Mortalin from HeLa cell extracts (lower panel). (D) Co-immunoprecipitation of endogenous RelA with Mortalin from U-2 OS and HEK293 whole cell extracts. Mouse IgG (mIgG) was used as a control in the IP. (E) In Rela −/− MEF cells reconstituted with RelA or a RelA T505A mutant, Mortalin was immunoprecipitated from whole cell lysates, followed by subsequent western blot. (F) Mitochondria were isolated from Rela −/− MEF cells reconstituted with RelA or RelA T505A mutant, followed by western blot analysis. (G) ChIP analysis of RelA binding to the D-loop region of the mitochondrial genome in Rela −/− MEF cells reconstituted with RelA or RelA T505A mutant (p<0.01, n=5/group).

Figure 6

The Heat Shock 70 family member Mortalin regulates RelA mitochondrial localisation. (A) Mortalin is required for RelA localisation in mitochondria. H1299 cells were treated with a mortalin siRNA and mitochondria were prepared. The presence of RelA in the mitochondria was determined by western blotting. (B) Knockdown of Mortalin in the reconstituted RelA MEF cells significantly reduced the binding of RelA to the cytochrome B (p<0.05, n=4/group) and D-Loop regions (p<0.05, n=3/group). (C) H1299 cells were treated with a pcDNA3 control or a pcDNA3-Mortalin expression plasmid, mitochondria were prepared followed by western blot analysis. (D) p53 inhibits the association of RelA with Mortalin. Co-immunoprecipitation of RelA, Mortalin and p53 was carried out in H1299^wtp53 extracts prepared with and without IPTG induction of p53.

Figure 7

Model of p53 and Mortalin dependent RelA regulation of cellular energy production. p53 can act to exclude RelA from mitochondria and together RelA and p53 promote oxidative phosphorylation. However, during tumorigenesis, upon loss of p53, RelA associates with Mortalin, enters mitochondria where it represses mitochondrial gene expression and thus oxidative phosphorylation. This then contributes to the switch to glycolysis observed in cancer cells.