p53 is a tumor suppressor protein that is central to genomic stability and is mutated in over 50% of all cancers.11
This molecule plays an important role in the cellular response to multiple types of stress including nucleotide depletion, hypoxia, oncogene activation or exposure to DNA damaging agents.11,12
Specific signaling pathways cope with genotoxic stress by initiating pauses in cell cycle progression to allow cells to survive and maintain themselves until the damage has been resolved or the stress has been removed.13
This is accomplished by cell cycle arrest, DNA repair, inhibition of ROS, angiogenesis through metabolic changes and autophagy. If the damage cannot be repaired, then multiple mechanisms can remove such cells via senescence, innate immune responses, apoptosis and tissue renewal. The activation and stabilization of p53 due to these multiple stress signals, depending on the amount of damage and tissue type, demonstrates the importance of p53 in many cellular functions. p53 has been directly implicated in activating these genotoxic pathways.14–18
All of these processes create a very complicated view of the functions of p53 in particular physiological contexts and specifically the pathways involving p21Waf1/Cip1
The p53/p21 pathway functions in determining cell arrest, apoptosis or senescence in response to genotoxic stress.15
was originally identified as an inhibitor of cyclin/cyclin-dependent kinases, a molecular complex necessary to proceed through the cell cycle. As a mediator of p53 in growth suppression, it is a marker for senescence in fibroblasts.19
is a member of the CIP/KIP family of CDK inhibitors that also include p27 and p57 that can inhibit a wide range of cyclin/CDKs to control progression through the cell cycle. The canonical function of the p21 protein is to direct cell cycle arrest in response to DNA damage in a p53-dependent manner.20,21
The process involves the binding of p21 to cyclin-CDKs, specifically cyclin D-CDK4/6 in G0
, preventing this complex from phosphorylating the retinoblastoma protein (pRb). Normally, hypophosphorylated Rb forms a transcriptional repressive complex with E2F that prevents expression of S phase-specific genes. Phosphorylation of pRb relieves transcription repression of E2F target genes, promoting cell cycle progression. p21 prevents Rb phosphorylation and maintains G1
arrest. The function of p21 in cell cycle arrest has also been extended to include S and G2
checkpoints through the interaction with PCNA and 14-3-3σ, respectively.22–24
In addition to the cell cycle checkpoint function of p21 in a p53-dependent manner, p21 also plays a direct p53-independent role in cellular senescence.25–27
Cellular senescence is defined as a permanent cell cycle arrest that can be triggered by an increase in reactive oxygen species, telomere shortening or by upregulation of an oncogene resulting in replicative stress.28
The function of senescence in cells appears to be a way of providing an obstacle to the progression of cancer by preventing damaged cells from undergoing aberrant proliferation.29–35
The two major pathways for activating senescence are controlled by p16Ink
or p53/p21, both of which lead to Rb hypophosphorylation. The p53-dependent senescence is directly modulated by p21 through detection of cellular stress; however, there have been other reports demonstrating that p21 can also elicit senescence in a p53-independent manner.36,37
For example, p21 was found to be essential in upregulating senescence-specific markers in cells that did not express p53.
A recent study18
examining hair follicle regeneration relates to our studies of ear hole regeneration. It was shown that p53 is an important component in the renewal of adult tissues that have “increased genomic instability phenotypes.”18,34,38
The activity of p53 is thought to be involved in clearing out cells that have accumulated DNA damage. This results in the induction of senescence and immune-mediated clearance.33
It is proposed that the accumulation of damaged cells that persist in adult tissue that fail to be cleared by p53-mediated mechanisms act as an obstruction to stem cell proliferation and tissue renewal.39
Thus, p53-induced senescence is required for tissue regeneration and the removal of p53 causes tissue renewal to become delayed due to the accumulation of damaged cells.
Given the discussion above, we asked several questions:
- If DNA damage and a DDR are seen and p53 is upregulated while p21 is downregulated as seen in the MRL mouse, what response should we expect? DNA damage should lead to p53 activation and an apoptotic response10,21 and not to senescence40 in the absence of p21. We actually found an increase in TUNEL-positive cells in regenerative MRL tissue, indicating that DNA damage is tolerated in these cells. We have not seen evidence for an increase in apoptosis in regenerative cells in culture.9
- upregulated in the MRL tissue, is p53 necessary to accomplish an ear hole closure regenerative response? To answer this, we crossed a p53−/− mouse41 to MRL producing first (MRLxp53+/−) F1 mutant mice, then MRLx(MRLxp53+/−) BC1 mutant mice, and then IC1 intercross mice producing WT, heterozygous and homozygous mutants. As seen in , 30 days after ear-punching (2 mm punch) of 8 week old mice, all female mice healed with a mean ear hole diameter ranging from 0.2−0.4 mm as is normally seen in parental MRL female mice.7 There were no statistically significant differences between WT (n = 11), heterozygous (n = 18) and homozygous p53 nulls (n = 5) even though the average homozygous null female hole size appeared smaller than the other two groups. The males showed larger hole sizes just on the border of the healing phenotype42 but, again, we found no significant differences in healing between any of the groups. Although more mice need to be analyzed, these early studies indicate that p53 is not essential for ear hole closure. It is clear from these results that lack of p53 does not have a negative effect on the regenerative ear hole closure response and may even have a beneficial effect. As discussed below (), MRL.p53−/− healing ear tissue displays interesting differences histologically from the MRL/MpJ healing ear tissue.
Figure 1 Mice were ear-punched using a 2 mm hole punch and hole diameter was read 2 and 4 weeks post-injury. Red columns = female mice, the blue columns = male mice; error bars show standard deviations. (A) MRL/MpJ mice were bred to p53−/− mice (more ...)
Figure 3 Histological analysis of ear sections from ear-punched mice 42 days post-injury. Ears were fixed and embedded,and sections through the hole were stained with Alcian blue (cartilage). In (A) sections at low magnification (4X) show the degree of healing (more ...)
- Since p53 is a key activator of p21, one question concerns why p21 is down in the MRL regenerator even though p53 is up. This would suggest that the MRL p53/p21 interaction is defective. Would elimination of p53 then lead to a regenerative response similar to that seen with the elimination of p21? To approach this question, we crossed heterozygous B6.p53 mutant mice43 and then ear-punched all offspring at 8–10 weeks of age. As shown below (), no significant differences were seen between the WT, heterozygous and homozygous p53 mutant mice with ear holes closing between 1.5–2.0 mm after 30 days, considered to be a non-healer phenotype.42 Interestingly, female mice, which are usually better healers than males, showed no significant differences.42
These results clearly demonstrate that p53 is not required for tissue and appendage (ear hole closure) epimorphic regeneration even in the case of high background levels of DNA damage as seen in the MRL mouse. This also suggests that p53-induced senescence and subsequent removal by the immune response is not essential for regeneration in the ear hole closure model. We have not ruled out p16-induced senescence, which is independent of p53 and p21. However, this typically is associated with the activation of an oncogene and we expect that this pathway is not involved in regeneration. We have shown that the p16-null transgenic mouse does not close ear holes () and we are breeding p21/p16−/− mice to further investigate the role of senescence in regeneration.
Figure 2 Preliminary analysis of mutant mice derived from MMHCC for regeneration capability. The ear pinnae of mice (n = 2 to 5) were wounded by hole punching and followed for 6 weeks. Healer (regeneration-competent) controls (MRL/MpJ and p21−/− (more ...)