p53 regulates mtDNA copy number and mitocheckpoint pathway

Abstract

Background: We previously hypothesized a role for mitochondria damage checkpoint (mito-checkpoint) in maintaining the mitochondrial integrity of cells. Consistent with this hypothesis, defects in mitochondria have been demonstrated to cause genetic and epigenetic changes in the nuclear DNA, resistance to cell-death and tumorigenesis. In this paper, we describe that defects in mitochondria arising from the inhibition of mitochondrial oxidative phosphorylation (mtOXPHOS) induce cell cycle arrest, a response similar to the DNA damage checkpoint response.

Materials and methods: Primary mouse embryonic fibroblasts obtained from p53 wild-type and p53-deficient mouse embryos (p53 -/-) were treated with inhibitors of electron transport chain and cell cycle analysis, ROS production, mitochondrial content analysis and immunoblotting was performed. The expression of p53R2 was also measured by real time quantitative PCR.

Results: We determined that, while p53 +/+ cells arrest in the cell cycle, p53 -/- cells continued to divide after exposure to mitochondrial inhibitors, showing that p53 plays an important role in the S-phase delay in the cell cycle. p53 is translocated to mitochondria after mtOXPHOS inhibition. Our study also revealed that p53-dependent induction of reactive oxygen species acts as a major signal triggering a mito-checkpoint response. Furthermore our study revealed that loss of p53 results in down regulation of p53R2 that contributes to depletion of mtDNA in primary MEF cells.

Conclusions: Our study suggests that p53 1) functions as mito-checkpoint protein and 2) regulates mtDNA copy number and mitochondrial biogenesis. We describe a conceptual organization of the mito-checkpoint pathway in which identified roles of p53 in mitochondria are incorporated.

Figure 1

Effect of mito-I on cell-cycle distribution of primary MEF. A) Treatment with mito-I, rotenone and TTFA-induced S-phase delay in a time dependent manner. Potassium-cyanide treatment causes delay in the S-phase after longer than 2 h treatment. B) Treatment with mito-I, antimycin oligomycin and CCCP induced S-phase delay in a time-dependent manner. The primary MEFs derived from p53 null mouse embryos treated with mito-I did not exhibit significant changes in cell-cycle progression

Figure 2

ROS production in primary MEFs treated with equimolar concentrations of mito-I. Cells were treated with equimolar concentrations of mito-I (10μM for 4h. A) p53-dependent production of H₂O₂ after treatment with antimycin, CCCP, potassium cyanide, rotenone and TTFA. Treatment with mito-I induced an increase in H₂O₂ production in primary MEF-expressing p53 but not in p53 null cells. B) No significant differences in O₂^- production between p53 +/+ and p53 -/- pMEF cells treated with equimolar concentration of CCCP, potassium cyanide and TTFA were found. Lowe panel shows p53-independent production of O₂^- after treatment with antimycin, oligomycin and rotenone

Figure 3

Mitochondrial damage modulates the level of p53 in primary MEFs. Western blot analysis of wild-type p53 cells treated with mito-I. Treatment initially leads to decreased expression of p53, but after six hours' exposure, the level of p53 becomes stabilized

Figure 4

Mito-I inhibition leads to translocation of p53 into mitochondria. A) Treatment of cultured HCT116 p53 +/+ cells for 24 hours with rotenone results in a concentration-dependent increase in p53 protein levels in the mitochondrial fraction. B) Quantitation of the p53 signal in mitochondrial fraction induced by rotenone. The intensity of the p53 and CoxII bands was measured by densitometry

Figure 5

Decreased mitochondrial DNA content and downregulation of p53R2. A) Decrease in mtDNA content in p53-/- cells compared to p53 +/+ cells. B) Down regulation of the p53R2 in p53-/- cells compared to p53 +/+ cells

Figure 6

Conceptual organization of mito-checkpoint response. Mitochondrial damage is recognized by sensor protein(s). The signal is mediated by mediator(s) (such as ROS, ATP) and transduced by transducer(s). The transducers regulate effectors (such as p53). Together, mito-checkpoint helps to maintain the mitochondrial integrity of cell