Main Research Findings

1) P21 plays a crucial role in treating cases of inflammatory arthritis of the synovium.

2) P21 was shown to help mediate stress responses and elicit a protective function against a wide variety of stressful stimuli.

 

Selected Data

1) The study conducted by the research team of Mavers et. Al examined how P21 limits the development of inflammatory arthritis as well as the induction of the wound healing phase that typically occurs after arthritic stimulation. The severity of inflammation and bone damage are related to the number of macrophages in the pannus. That being said, the C-terminal domain of the P21 peptide has been shown to have the ability to reduce the amount of macrophages in the pannus. The study included male KRN mice, obtained from Dr. Diane Mathis, which were crossed with female NOD mice, purchased from Taconic in Hudson, NY. The mice were backcrossed onto the C57B1/6 background for approximately 12 generations and tested for microsatellite markers [1].

Serum was harvested from 8 week old KRN and NOD mice via cardiac puncture, followed by an intraperitoneal injection into 6-8 week old backcrossed mice. A caliper was used to calculate ankle circumference of the test subjects; results were scored as follows: 0=no swelling, 1=mild in<3 limbs, 2=mild in >2 limbs, 3=moderate in >2 limbs, 4=severe in >2 limbs, and 5=compromised mobility. The test subjects were euthanized 7, 14, or 25 days post-injection followed by serum collection and ankle harvesting. The ankles were then subjected to microCT and/or prepared for further immunohistochemistry analysis.

The Wt mice were then intraperitoneally injected with 10 mg/kg of P21, 30 minutes before K/BxN serum and daily throughout the experimental period. 3 days after intraperitoneal injection of 4% aged thioglycolate adhered in serum-free media and maintained in DMEM, peritoneal cells were harvested from 6-8 week old male and female mice via lavage. Peritoneal macrophages were then incubated with 50 uM of the peptide for 2 hours followed by LPS stimulation in the presence of P21 [1].

2) The research team of Gorospe et. Al examined the functional role that the peptide P21 plays in response to stress. Particular genes are categorized as stress-response genes and are typically activated in response to various types of stressful stimuli. While the exact mechanism is still unclear, there is evidence supporting the theory that stress-response genes are involved in the determination of a cell’s fate. The peptide P21 plays a crucial role in the regulation of cell growth and the expression of this gene tends to be highly upregulated in the presence of stressful stimuli [2].

In terms of the transcriptional regulation of P21, recent studies have found that many transcription factors are implicated in the regulation of P21 expression. P21 promoter sequences reveal the conservation of at least two P53 binding sites. This is important to mention considering that the presence of P53 contributes to the magnitude of P21 expression. In cases of ionizing radiation P53 is actually required in order to induce the expression of P21. Additionally, the P21 promoter sequence contains six SPI binding sites in the proximal region. Different sites are required for binding each member of the multigene familiar when under different stress conditions. Enhancing the expression of P21 through different stressful stimuli can also be regulated by altering the stability of the P21 mRNA. The normal half-life of P21 mRNA is approximately 1 hour, however, exposure to stressors such as gamma-irradiation, TNF-alpha, or diethyl-maleate, increases the half-life to greater than 4 hours [2].

It was recently reported that UVC light induces P21 expression through a posttranscriptional mechanism that involves the stabilization of P21 mRNA; the induction of this mechanism is dependent on the presence of functional P53 considering that the P53 protein has the potential to bind RNA and regulate mRNA stability. More recently it was found that the compound HuD, a neuronal-specific protein, binds in a site-specific manner with high affinity to P21 mRNA. P21 mRNA also binds to a similar compound, HuR. Formation of the P21-HuR complex was shown to be inducible by UVC irradiation. Evidence has shown that cells that express lower levels of HuR exhibit reduced binding to P21 mR, diminished P21 mRNA inducibility after UVC treatment, and a shortened P21 mRNA half-life. This suggests that HuR is required for the induction of P21 through UVC while the stabilization of P21 mRNA tends to be regulated by HuR [2].

 

Discussion

1) The results of the study conducted by Mavers et. Al reported that there were no differences in the total number of leukocytes or the populations of leukocytes in the P21 knockout mice backcrossed onto the C57B1/6 background. The P21 knockout mice developed significantly worse ankle swelling in comparison to the control wild type mice. Ankle swelling was determined by measuring the ankle circumference followed by an intraperitoneal injection of K/BxN serum in order to examine the development of inflammatory arthritis. The P21 knockout mice also displayed a 3-fold evidence of disease severity that was the most pronounced at day 25. The disease resolved in the Wt control mice but not in the mice with the P21 gene knocked-out [1].

 

Figure 1: Changes in ankle circumference after intraperitoneal serum injection

The ankles of both Wt and P21 knockout mice were harvested and histologically analyzed 7, 14, and 25 days after K/BxN serum was injected. At days 7 and 14 the researchers observed inflammation and pannus development in the joints of both Wt and P21 knockout mice. While pannus formation was most notable at days 7 and 14 it is important to mention that there was a detectable pannus at all time points in the P21 knockout mice. Additionally, formation of the pannus correlated to enhanced destruction of cartilage at all points in time in the P21 knockout mice. However, at day 14 there was a 3-fold increase in cartilage destruction in comparison to the Wt control mice. When comparing the P21 knockout and the Wt mice, the research team observed increased numbers of TRAP-positive cells localized in the pannus, as well as increased bone damage in the P21 knockout mice as late as 25 days post-injection despite no arthrogenic antibodies present. These findings indicate that deficiency in P21 leads to inflammation and/or a failure to resolve the inflammation [1].

The increased inflammation and bone destruction was further examined through hematoxylin and eosin stained sections as well as analysis of the infiltration of immune cells into the arthritic joints. The ankle sections were stained for CD45 hematopoietic cells, F4/80 macrophages, and proliferation cell nuclear antigen (PCNA). On day 7 and 14 the amount of CD45 hematopoietic cells increased by 2.8-fold in the pannus and 1.4-fold in the synovium of the ankles of P21 knockout mice. However, by day 25 there was no difference in the number of hematopoietic cells when comparing the P21 knockout mice and the Wt control mice.

The P21 knockout mice experienced peak PCNA staining in the ankles at day 7 post-injection, while the Wt control mice experienced peak PCNA staining at day 14. This indicates that there is likely increased proliferation of synovial fibroblasts as macrophages are terminally differentiated. By day 25 post-injection there was no difference in peak PCNA staining in P21 knockout or Wt mice, suggesting that synoviocytes could potentially play a vital role in the early stages of disease development and infiltration of inflammatory cells. When quantifying the amount of macrophages in the pannus, the P21 knockout mice experience a 3.7-fold increase at day 7, 1.6-fold increase at day 14, and a 1.8-fold increase at day 25. Since there were more macrophages than CD45-positive cells by day 25, the researchers were able to conclude that P21 knockout mice could potentially not be able to resolve arthritis due to the macrophages in the synovium [1].

Figure 2: changes in the number of positive cells in both Wt and P21 knockout mice

Next, the researchers examined the impact of delivering established domains of P21 to arthritic mice in an attempt to better understand the critical domain of P21 that plays a crucial role in inflammatory suppression. Previous research has suggested critical interactions between domains on P21 and cyclins, CDKs, and PCNA, and how the actions of these compounds result in altered cell cycle progression. 5 sets of P21-peptide mimetics were then developed and conjugated to a polycationic peptide derived from the HIV-1 transactivator of transcription that allows cell entry. The functionality of these peptidomimetics were then evaluated in regards to the suppression of K/BxN serum transfer arthritis. When treated with amino acids 141-160, the mice experienced a 36-fold reduction in ankle swelling by day 2, a 6-fold decrease by day for and a 4-fold reduction by day 7. These mice also exhibited a marked enhancement in clinical scoring in comparison to control groups. Any changes observed in the subjects treated with amino acids 15-40 or 63-70 were deemed insignificant [1].

Figure 3: Changes in ankle circumference and clinical scores depending on days post-serum transfer.

Seven days after arthritis was induced and peptide treatment was administered, the ankles of the test subjects were harvested and histologically examined. Treatment with the peptide corresponding to amino acids 141-160 consistently led to a dramatic decrease in inflammation, pannus development, synovial lining thickness, infiltration of lymphocytes, and bone erosion. Additionally, there was decreased infiltration of the hematopoietic cells and macrophages within the pannus of the mice treated with amino acids 141-160. However, there were no differences detected in the normal synovium or the level of cartilage destruction. PCNA staining also found that several of the peptide treatments administered to the mice led to reduced cellular proliferation throughout the pannus [1].

Figure 4: Ankle sections scored on a scale of 0-5 by a pathologist blinded to the study

2) The results of the study conducted by Gorospe et. Al examined how P21 expression is regulated by stress in order to understand the role of the peptide during the stress response. Two opposing views first emerged arguing that either P21 played a role in the implementation of apoptosis, or that P21 prevents apoptosis by serving a protective function. Justification for the first argument was based on the fact that P53-regulates P21, and P53 is highly related to apoptosis. The second argument hypothesizes that the expression that P21 is crucial to the growth arrest that happens following stressful stimulation. This process is important as it allows the cell the time to repair any stress-induced damages before any DNA is passed to daughter cells. Most published research supports the hypothesis that P21 plays a protective role against cellular stress. However, the effects of P21 can potentially vary; there are situations where P21 has no significant benefit for the cell and others where increased P21 levels are detrimental to the cell [2].

Figure 5: Schematic representation of the influence P21 has on cell survival

Additionally, the results of the study observed the lack of detectable consequences of P21 on cellular outcome during stress. In the reported examples where the peptide failed to modulate cell survival after stressful stimuli, researchers saw that P21 did not alter survival rates of the PC 12 rat pheochromocytoma cells that had been previously subject to withdrawal-induced apoptosis. P21 expression was not found to enhance or reduce occurrences of withdrawal-induced cytotoxicity, however, it’s important to mention that serum withdrawal is one of the few stressful stimuli that typically decreases rather than increases the expression of the P21 peptide.

P21 was also found to fail when exhibiting a detectable influence on cellular outcome in terms of understanding the cell’s response to oxidative stress. By exposing HeLa cells to hydrogen peroxide, there was a minor increase in the expression of P21 that was significantly influenced by narrow time- and dose-dependent limits. The HeLa cells were then either mock-infected or infected with an adenovirus. Both were treated with hydrogen peroxide and the rate of apoptosis was examined at varying times following the treatment. When P21 is overexpressed in the mock-infected cells, the peptide is able to protect against H2O2-mediated apoptosis, while the infected cells experienced high levels of toxicity and apoptosis after H2O2 treatment [2].

Figure 6: Changes in the percent of apoptotic cells in response to the presence of the adenovirus

As it was previously mentioned, P21 can occasionally have a detrimental influence on the cells following the presence of a stressful stimulus. One of the first examples revealed that ectopic overexpression of P21 in MCF7 or T47D cells resulted in significant levels of apoptosis, primarily associated with the formation of giant cells and the process of growth arrest. A similar mechanism was observed in U87 glioma cells. Transfection with a P21 expression vector resulted in increased levels of cell death, however, the same could not be said for various other glioma cell lines, such as GB-1. Overexpression of P21 did not directly lead to cell death, however, it allowed the cells to become sensitized to cisplatin-induced apoptosis. The current study found that while there were limited occurrences of detrimental P21 expression, infection of Rat1 cells with Ad.p21 resulted in extreme cytosis and rapid apoptosis of the infected cells. The researchers suggested that P21 was responsible for this response considering that Ad.null-infected cells failed to exhibit the same toxic response [2].

Figure 7: The percentage of remaining Rat1 cells after infection with Ad.p21.

Overall, researchers Gorospe et. Al were able to conclude that despite the tight regulation of P21 expression during the stress response, further research still needs to be conducted regarding the mechanism through which the peptide influences cellular outcome. The data revealed that P21 typically performs a protective function within the cells, however, it was suggested that the beneficial role of the peptide is related to its ability to induce growth arrest through its ability to inhibit the effects of cyclin-dependent kinases. The research team suggested analysis of the P53 protein should be carried out in order to fully understand its apoptotic, transcriptional, and growth-inhibitory functions, as well as the mechanisms involved and the resulting effects on other cells. That being said, the researchers determined it would also be helpful to conduct a separate analysis of the various domains of P21 and how they interact with cdks and PCNAs. This method also has the potential to answer whether the growth-inhibitory function of P21 can be separated from its protective functions [2].

 

Disclaimer

**LAB USE ONLY**
*This information is for educational purposes only and does not constitute medical advice. THE PRODUCTS DESCRIBED HEREIN ARE FOR RESEARCH USE ONLY. All clinical research must be conducted with oversight from the appropriate Institutional Review Board (IRB). All preclinical research must be conducted with oversight from the appropriate Institutional Animal Care and Use Committee (IACUC) following the guidelines of the Animal Welfare Act (AWA).

 

Citations

[1] Mavers M, Cuda CM, Misharin AV, Gierut AK, Agrawal H, Weber E, Novack DV, Haines GK 3rd, Balomenos D, Perlman H. Cyclin-dependent kinase inhibitor p21, via its C-terminal domain, is essential for resolution of murine inflammatory arthritis. Arthritis Rheum. 2012 Jan;64(1):141-52. doi: 10.1002/art.33311. PMID: 21898359; PMCID: PMC3253189.

[2] Gorospe M, Wang X, Holbrook NJ. Functional role of p21 during the cellular response to stress. Gene Expr. 1999;7(4-6):377-85. PMID: 10440238; PMCID: PMC6174658.

 

P21 is also known as cyclin-dependent kinase (CDK) inhibitor 1. It is involved in the processes of apoptosis and cell cycle arrest and has been shown to block the activity of cyclin and CDK complexes in animal modules.

A study conducted by Mavers et. Al examined the potential P21 has in use relating to synovial inflammation that is consistent in animals with rheumatoid arthritis. In a previous study, Mavers et. Al found that in animals with rheumatoid arthritis there is decreased expression of P21 throughout the synovium, leading them to conclude that P21 has a suppressant effect on the inflammatory responses of the macrophages. This alludes to the idea that in the presence of P21 there will be less inflammation and tissue breakdown in the synovium.

The researchers of the initial study induced arthritis in mice that were lacking the expression of P21. From there, the next step was to treat the P21 knockout mice with a P21 mimetic which acted as a prophylactic for the development of arthritis in the mice. In the mice treated with P21 mimetic, the use of Luminex-based assays, flow cytometry, and ELISAs were used in order to observe LPS-induced cytokines and the signal transduction pathways throughout the macrophages in order to determine the strength of the inflammatory response in the presence of the P21 mimetic.

The results of this study showed that in the mice lacking the presence of P21 had drastic development of arthritis. However, in the mice treated with the P21 mimetic the activation of the macrophages was inhibited. Since there was a reduced inflammatory response in the macrophages which led to exacerbated arthritis symptoms, the researchers concluded that the presence of P21 could potentially play a key role in treating rheumatoid arthritis (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3253189/).

 

Effects of P21 on Alzheimer’s Disease and Cognitive Defects

The integrity of dendritic spines is essential to proper cognitive functioning. Mental delays are frequently caused by mutations in the P21-activated kinases (PAK) due to the important role that PAK-cofilin signaling plays in the morphogenesis of the dendritic spines and regulation of actin filaments. A study conducted by Zhao et. Al examined how the PAK pathway could potentially be involved in the development of Alzheimer’s disease. It was found that in cases of Alzheimer’s Disease PAK activity was remarkably reduced which goes hand in hand with decreased phosphoPAK, pathology of cofilin proteins, and loss of the protein drebrin which is an actin-regulatory protein.

Additionally, the study found the 𝜷-amyloid (A𝜷) was directly involved in drebrin loss and defects in PAK signaling pathways in hippocampal neurons that were treated with A𝜷. Furthermore, the same defects were found in Appswe transgenic mice that possessed a double mutation leading to overproduction of A𝜷. It was also noted that in adult mice, inhibition of PAK signaling led to similar instances of cofilin pathology, loss of drebrin protein, and memory loss that is also seen in cases of Alzheimer’s disease. The findings of this study indicate that activation of the P21-activated kinase pathway can potentially protect against Alzheimer’s as well as various other forms of declining cognitive functioning (https://www.nature.com/articles/nn1630).

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