The role of p21 in cellular senescence and aging-related diseases

Abstract

During the aging process or disease progression, normal cells and tissues in the body undergo various stresses, leading to cell damage and the need for repair, adaptation, apoptosis, or defense responses. Cellular senescence is a key player in this process, influencing the rate of aging and disease progression. It can be triggered by different stress factors, resulting in irreversible cell cycle arrest and functional decline. Senescent cells often show high expression of cell cycle factors such as p21 and p16, which are involved in cell cycle arrest. p16 has long been recognized as a significant marker of aging. Recent evidence suggests that p21^high cells and p16^high cells represent distinct cell populations in terms of cell type, tissue location, accumulation kinetics, and physiological functions. This article focuses on recent advancements in understanding p21-dependent cellular senescence. It starts by providing an overview of the role of p21 in 3 primary cellular senescence phenotypes where it plays a crucial role. It then delves into the pathogenesis of diseases closely linked to p21-dependent cellular senescence, particularly metabolic disorders and cardiovascular diseases. The article also discusses progress in p21-related animal models and outlines strategies for utilizing p21 to intervene in cellular senescence by delaying aging, eliminating senescent cells, and rejuvenating senescent cells. This review systematically examines the pathogenesis of p21-dependent cellular senescence, emphasizing its importance in studying aging heterogeneity and developing new senolytic therapies. It aims to stimulate future research on leveraging p21 to enhance the characteristics of senescent cells, allowing more precise methods for eliminating harmful senescent cells at the right time, thereby delaying aging and potentially achieving rejuvenation.

Highlights

• •P21^high and p16^high cells are distinct cell populations with different types, locations, accumulation kinetics, and functions.
• •We propose strategies to use p21 for intervening in cellular senescence by delaying aging, eliminating senescent cells, and rejuvenating them.
• •Understanding the pathogenesis of p21-dependent senescence is crucial for studying aging heterogeneity and developing senolytic therapies.
• •Encourage future research on utilizing p21 to improve senescent cell characteristics.

INTRODUCTION

P21 is a member of the Cip/Kip family of cyclin-dependent kinase (CDK) inhibitors, also referred to as p21 WAF1/Cip1 or CDK inhibitor 1A. The tumor suppressor gene p21 was first identified by El-Deiry et al. (1993), and it plays a significant role in inhibiting tumor proliferation in brain, lung, and colon tumor cells. This gene is located on the short arm of chromosome 6, downstream of the p53 gene. The protein encoded by this gene consists of 165 amino acids (Romanov and Rudolph, 2016).

The function of p21 is multifaceted, playing key roles as a cell cycle inhibitor, senescence inducer, tumor suppressor, and regulator with a broad range of activities (Romanov and Rudolph, 2016). Beyond these roles, p21 is also crucial in inducing apoptosis, differentiation, reprogramming pluripotent stem cells, DNA repair, transcription, and cell migration (Kreis et al., 2015). There is ongoing discussion regarding its involvement in cellular senescence. It is well-established that the Cip/Kip family, including p21, can inhibit cyclins A, D, and E, thereby preventing cells from advancing to subsequent stages of the cell cycle, ultimately leading to cellular senescence (El-Deiry, 2016, Kreis et al., 2016).

p21 is a well-established cell cycle inhibitor, playing a crucial role in halting cell cycle progression at the G1/S and G2/M transitions (Bertoli et al., 2013). In the G1 phase, p21 suppresses the activity of the cyclin D-CDK4/6 complex, which facilitates DNA damage repair. During the G1 to S phase transition, p21 primarily induces G1 arrest by inhibiting the actions of cyclin E/CDK2 and cyclin A/CDK2. This inhibition results in the hypophosphorylation of pRB, thereby preventing the release of E2F transcription factors and blocking the cell cycle's entry into the S phase. Furthermore, p21's inhibition of cyclin B/CDK1 activity can lead to cell cycle arrest at both the G2 and G2/M phases (Chen et al., 1996).

The p21-regulated cell cycle pathway operates through both p53-dependent and p53-independent mechanisms. In the p53-dependent pathway, exposure to factors such as DNA damage or oxidative stress triggers an increase in p53 activity. The p53 protein then binds to a specific response element in the p21 promoter region, leading to the activation of p21 expression. This activation inhibits cell cycle progression, allowing time for DNA repair (Jung et al., 2010). If the DNA is successfully repaired, the cell can proceed to the S phase. However, if the DNA repair fails, the BCL-2 family protein BAX is activated, initiating apoptosis and contributing to the apoptotic pathway. In the p53-independent pathway, p21 expression can be regulated by various upstream factors, such as c-Myc (Palazzo et al., 2016, Pavlides et al., 2016, Wu et al., 2003).

p21 has emerged as a key focus in cellular senescence research, with growing evidence indicating that p21^high cells constitute a distinct population of senescent cells, separate from those associated with p16. This article offers a comprehensive overview of 3 senescence phenotypes involving p21, elucidates the differences between p21 and p16, and examines the role of p21-dependent senescence in the pathogenesis of various diseases. The article also reviews the progress in research using p21-related animal models. Finally, it explores the therapeutic potential of targeting p21, particularly in clearing senescent cells, slowing the aging process, and rejuvenating senescent cells.

THE ROLE OF P21 IN CELLULAR SENESCENCE

The concept of cellular senescence was first introduced by Hayflick and Moorhead in 1961, when they observed that proliferating human diploid fibroblasts could enter a state of irreversible cell cycle arrest while still maintaining metabolic activity (Hayflick and Moorhead, 1961). Cellular senescence is a nonproliferative state triggered by various stressors, such as oxidative stress, DNA damage, and inflammation, often occurring during aging or disease progression. Cells primarily enter senescence through 2 key pathways: the p16/Rb pathway and the p53/p21 pathway (Khosla et al., 2020). The phenotypes of cellular senescence are diverse and can be categorized into 8 types based on different damaging stimuli (Hernandez-Segura et al., 2018). This review specifically focuses on 3 senescence phenotypes that are currently associated with p21.

Replicative Senescence

Replicative cellular senescence is characterized by a gradual decline in a cell's ability to divide after multiple rounds of replication, eventually leading to a complete halt in cell division. This phenomenon occurs because each replication cycle shortens the telomeres, which, in most cells, cannot be regenerated or extended. As a result, telomere attrition imposes a limit on the cell's proliferative capacity (Hernandez-Segura et al., 2018). Notably, p21 has been identified as a key player in this senescence phenotype.

p21 plays a dual role in the cell cycle, with its effects being largely dependent on its expression levels. Elevated levels of p21 inhibit the cyclin D1-CDK4 complex, leading to cell cycle arrest. Conversely, lower levels of p21 promote the formation of the active cyclin D1-CDK4 complex, thereby facilitating cell proliferation (Abbas and Dutta, 2009). Furthermore, the regulation of cell proliferation by p21 is closely linked to the expression dynamics of Hes1. Research indicates that oscillating expression of Hes1 suppresses p21, whereas continuous overexpression of Hes1 leads to upregulation of p21. As a result, Hes1 plays a critical role in controlling neural stem cell proliferation via p21 (Maeda et al., 2023). While the p53-p21 axis is well-established as a key pathway for inducing cell cycle arrest, p21 also regulates this process through p53-independent mechanisms. For example, in long-term cultured astrocytes, the transcription factor old astrocyte specifically induces substance/CREB3L1 directly triggers p21-mediated cell cycle arrest at the G2/M phase. This action results in compromised DNA integrity due to repeated replication cycles, highlighting old astrocyte specifically induces substance as a cell cycle inhibitor through p21, with potential implications for regulating cell aging (Saito et al., 2023).

Developmentally Programmed Aging

Cellular senescence can occur as early as the embryonic period, indicating its role in developmental programming. This type of senescence is characterized by the absence of detectable DNA damage and the lack of activation of ataxia telangiectasia mutated and ataxia telangiectasia and Rad3-related pathways. Furthermore, this form of senescence is p21-dependent and is essential for proper embryonic development and tissue formation (von Kobbe, 2018).

Before embryo implantation, the protein p21 plays a pivotal role in regulating cellular senescence. Studies have shown that exposure of embryos to heat stress or radiation increases p21 expression, and reducing p21 mRNA levels can alleviate cell cycle arrest (Ock et al., 2020). As a key regulator of the cell cycle, p21 is particularly important for placental development. In early pregnancy, p21 is highly expressed in the placenta, with its expression significantly influenced by oxygen levels. Hypoxia, or low oxygen conditions, can decrease p21 expression in trophoblast cells, thereby impairing their ability to fuse. Consequently, disruptions in p21 levels may disturb cellular balance and potentially elevate the risk of preeclampsia (Kreis et al., 2021).

p21 is also essential in the proliferation of embryonic stem cells. It is directly regulated by ten-eleven translocation 1 (Tet1), which influences the pluripotency, proliferation, and differentiation of these cells. Tet1 reduces the levels of EZH2 and H3K27 trimethylation at the p21 promoter, thereby facilitating the rapid transition from the G1 to the S phase of the cell cycle. This transition is a crucial marker of embryonic stem cell proliferation (Chrysanthou et al., 2022).

p21 is a critical factor in tissueogenesis, particularly in coordinating endothelial cell proliferation during vascular formation and growth. Research indicates that a mutation in the RNA helicase Ddx21 triggers Vegfc-Flt4-driven endothelial cell proliferation. In the absence of Ddx21, p21 expression increases, leading to cell cycle arrest and the inhibition of lymphangiogenesis. This mechanism may be relevant in diseases characterized by excessive lymphangiogenesis (Koltowska et al., 2021). Additionally, p21-dependent cellular senescence plays a vital role in normal neural development. The loss of Rack1 activates the p21-dependent signaling pathway, inducing an aging phenotype in neural stem cells. Remarkably, removing p21 can effectively mitigate the microcephaly phenotype observed when Rack1 is knocked out in vivo, suggesting that Rack1 suppresses p21-induced neural stem cellular senescence—a crucial mechanism for proper neural development (Zhu et al., 2021). Furthermore, p21 is essential in the formation of the endocardial cushion, with studies revealing that β-catenin inhibits p21 to promote cell proliferation. This indicates that the β-catenin/p21 axis may play a role in the development of congenital heart defects (Liu et al., 2023).

DNA Damage Induces Cellular Senescence

Irreversible DNA damage, such as that caused by radiation and various drugs, can induce cellular senescence (Hernandez-Segura et al., 2018). The role of p21 in regulating cellular senescence under stress conditions has been well documented. For example, the ARF-interacting protein collaborator of ARF (CAFR)/CDKN2AIP has been shown to induce cell growth arrest and senescence through the ARF-p53-p21 signaling pathway. The expression level of CAFR is considered a reliable quantitative predictor of cell proliferation fate (Kalra et al., 2023). Moreover, cellular senescence resulting from different types of DNA damage exhibits heterogeneity, meaning that cells exposed to the same dose of DNA damage may have varied outcomes. Studies have demonstrated that p21 can kinetically influence whether cells continue mitosis under radiation stress, as well as affect the division rate. Therefore, p21 serves as a more precise monitor to evaluate DNA damage and potentially predict long-term cell fate (Tran et al., 2023). Interestingly, while p21 does not play a significant role in an unperturbed cell cycle, it is crucial in coordinating cell cycle arrest under stress conditions to help cells adapt. Research shows that when stressors activate the phosphatase calcineurin, they can extend Hog1-dependent phosphorylation of p21, leading to the downregulation of G1 cyclin transcription and triggering G1/S arrest. This suggests that p21 assists cells in prolonging cell cycle arrest, allowing them more time to adapt to new environmental conditions (Flynn and Benanti, 2022).

COMPARISON BETWEEN P21 AND P16

Both p21 and p16 are involved in the cell cycle arrest of senescent cells, but they function through distinct mechanisms. The p16 gene product, p16INK4, competes with cyclin D to bind CDK4/6, inhibiting the phosphorylation of the RB protein and blocking the transition from the G1 phase to the S phase. Additionally, studies have shown that p16 can promote the degradation of phosphorylated RB protein, further inhibiting cell cycle progression (Aprelikova et al., 1995, Min et al., 2014, Haferkamp et al., 2008). In contrast, p21 inhibits CDKs A, D, and E, preventing cell cycle progression at the G1/S and G2/M transitions (Huang et al., 2022, Romanov and Rudolph, 2016). Beyond their differences in cell cycle arrest pathways, p16 and p21 also vary in terms of their associated senescent cell subpopulations, stress regulation mechanisms, secretory phenotypes, and impacts on lifespan (Fig. 1).

Fig. 1.

Comparison of p16 and p21 in cellular senescence.

Heterogeneity of Cellular Senescence

Increasing evidence indicates that p21^high cells and p16^high cells represent distinct subpopulations of senescent cells, which are found in different tissues. p16 is predominantly expressed in pancreatic β cells (Helman et al., 2016), bone cells (Doolittle et al., 2023), lung cells (Demaria et al., 2014, Kaur et al., 2021, Reyes et al., 2022), and other cell types. In contrast, p21 is primarily expressed in adipocytes (Choudhery et al., 2014, Wang et al., 2020, Wang et al., 2022), callus at fracture sites (Chandra et al., 2020, Chandra et al., 2022), lung fibroblasts (Chen et al., 2020, Jiang et al., 2017, Zhong et al., 2022), and trophoblast cells in the placenta (Cindrova-Davies et al., 2018, Higuchi et al., 2019, Song et al., 2021, Velicky et al., 2018 ).

The accumulation of p21 in adipose tissue is closely linked to tissue dysfunction associated with age-related diseases. A single-cell transcriptomic study revealed that p21^high cell populations in adipose-derived mesenchymal stem cells (AMSCs) from aged mice exhibit characteristics of senescence and reduced regenerative potential (Choudhery et al., 2014, Wang et al., 2020). Moreover, in both natural aging and obesity, p21^high cell accumulation is more prevalent than p16^high cell accumulation (Wang et al., 2021, Wang et al., 2022). This suggests that p21 plays a significant role in the heterogeneity of aging within adipose tissue.

Stress and Regulatory Mechanisms

p21 is notably expressed early in the acute phase of traumatic injury. For example, following a mouse fracture, p21 levels in the callus initially increase, followed by a rise in p16 and senescence-associated secretory phenotype (SASP) (Chandra et al., 2020). Similarly, in bone aging induced by focal radiation therapy, p21 is prominently expressed at early stages, whereas p16 appears later. Early targeting p21-positive cells, as opposed to p16-positive senescent cells, can mitigate bone loss caused by focal radiotherapy (Chandra et al., 2022). These findings highlight the functional heterogeneity between p21^high and p16^high senescent cell subpopulations and underscore differences in their regulatory stages. Early intervention targeting p21 may yield more effective results.

Secretory Phenotypes

Senescent cells secrete proinflammatory cytokines and other factors, a hallmark known as the SASP. This phenomenon disrupts tissue homeostasis and induces a proinflammatory state (de Magalhães and Passos, 2018, Zhao et al., 2023). The components of SASP can vary depending on the disease context and senescence inducers. Among these, bioactive secretions related to p21 are referred to as the p21-activated secretory phenotype (PASP) (Hu et al., 2024, Sturmlechner et al., 2021). p21 is involved not only in cell cycle arrest of senescent cells but also in early-stage immune surveillance of stressed cells. It aids in clearing stressed cells, and when the stress surpasses the cell's repair capacity, p21 releases chemokines such as CXCL14 to recruit macrophages and maintain multicellular environment homeostasis (Sturmlechner et al., 2021). Research indicates that PASP components differ from SASP components and change dynamically over time. This suggests that the differences in secretion phenotypes between p21 and p16 may be a key factor in their distinct biological functions.

Effects on Aging and Lifespan

p21^high and p16^high cells have different effects on aging and lifespan. Drugs targeting senescent cells, such as dasatinib and quercetin, can extend the median lifespan of mice by eliminating p16^high cells, but they do not affect the maximum lifespan (Baker et al., 2016). In contrast, intermittent depletion of p21^high cells not only extends the natural lifespan and reduces the risk of death in aged mice but also significantly improves physical function and health at every stage of the life cycle. Deletion of p16^high cells, on the other hand, does not enhance healthspan and may actually reduce health in later years (Wang et al., 2024).

P21-DEPENDENT CELLULAR SENESCENCE IN AGING-RELATED DISEASES

Cellular senescence is linked to various noncommunicable diseases (Chaib et al., 2022). However, a universal marker for identifying senescent cells remains elusive. p21 has emerged as a promising candidate for this purpose (Gasek et al., 2021, Gorgoulis et al., 2019). Research has examined the number of p21^high cells in tissue arrays and their correlation with age, revealing that the number of p21^high cells increases with age in the skin, pancreas, and kidney, while it remains stable in the lungs. Notably, no p21^high cells are observed in muscle tissue (Idda et al., 2020). Overall, the relationship between p21^high cell counts and age varies across different organs, exhibiting both beneficial and detrimental effects. The following section will delve into the role of p21^high cells in the context of various diseases (Fig. 2; Table 1).

Fig. 2.

p21-dependent cellular senescence in aging-related diseases.

Table 1.

p21-dependent aging-related diseases

Disorders and diseases	Genetic model	Pharmacologic agent	Phenotype of intervention	Pathway	Significance	References
Osteoporosis	p21-ATCC, p16-ATCC, cyclophilin B–deficient mice	AP20187, LMV, senolytics	Improve osteoporosis and bone marrow obesity, restore bone formation.	Sirt1/p53/p21 axis	p21 dominates radiation-induced osteoporosis, and p21 inhibitors have osteogenic potential.	Chandra et al. (2022) Wen et al. (2021) Zhang et al. (2022)
Osteoarthritis	p21-KO, CCN3-KO, Sirt1-KO	Recombinant CCN3 protein, p53 inhibitors	Aging, apoptosis, and hypertrophy of chondrocytes lead to cartilage degeneration.	p53/p21	Chondrocyte p21-dependent cellular senescence is a potential therapeutic target for osteoarthritis.	Song et al. (2021) Takashima et al. (2021) Kuwahara et al. (2020) Xu et al. (2020)
Metabolic disease	UCP1-RFP, SPRY1 KO ASCs, p21-Cre, db/db mice	ROS scavenger N-acetyl-l-cystein, dasatinib plus quercetin, 3K3A-aPC and parmodulin-2	Adjust fat growth differentiation, improve obesity and diabetes.	SIRT1/p53/Ac-p53/p21 axis	p21 has great clinical application prospects in metabolic disorders.	Park et al. (2021) Xiang et al. (2020) Mandl et al. (2020) Wang et al. (2022) Al-Dabet et al. (2022) Oh Sung et al. (2023)
Damage repair	tet-p21, p21-KO	High-concentration trehalose, γ-secretase inhibitor, lopinavir, and/or ritonavir	p21 affects fracture healing, joint injury repair, lung injury, and liver healing.	Ihh-Runx2-Osterix, p53/p21, Notch-sirtuin 1-p21	p21-dependent aging occurs instantaneously to accelerate damage repair, and the medication time needs to be discussed to ensure effectiveness.	Chia et al. (2021) Kozyrska et al. (2022) Brauer et al. (2023) Muto et al. (2023) Ibaraki et al. (2020) Kikuchi et al. (2022) Blázquez-Prieto et al. (2021) Duan et al. (2022)
Pulmonary disease	BLM-ILD	Senolytics	p21-dependent cellular senescence promotes pathological progression of COPD and pulmonary fibrosis.	p53/p21	Developing new treatments targeting p21+ senescent cells or SASP molecules.	Yang et al. (2022) Levi et al. (2023) Lv et al. (2022) Yamada et al. (2022)
Neuropathic disease	SATB1-KO	Harmine	p21-dependent senescence is a pathological contributor to Parkinson's disease, Huntington's disease, and spinal atrophy.	p53/p21	Therapeutic strategies targeting p21 may be a beneficial avenue to intervene in some neurological diseases.	Riessland et al. (2019) Habib et al., (2022) Reedich et al. (2021)
Cardiovascular disease	Kawasaki vasculitis mouse model, p21-KO, spontaneously hypertensive rats, miR-26a–deficient mice, high-fat-diet (HFD)-fed ApoE−/− mice, Lrp6-deficient mouse, miR-499-5p-overexpressing mice	Rapamycin, sirt1, p21-adenovirus and p21-siRNA, oridonin, calcitriol, microRNA-26a, rivaroxaban, AAV9 miRNAi-Lrp6 construct, lncRNA-p21 or si-lncRNA-p21, miR-499-5p, folic acid	p21 plays an important role in the occurrence and development of cardiovascular diseases, such as cardiac hypertrophy, myocardial fibrosis, atherosclerosis, myocardial infarction, and heart failure.	p53/p21, p21/LC3B, Sirt1/p21, NF-κB, ING5/P21, Wnt/β-catenin, miR-499-5p-p21 axis	p21-dependent cellular senescence is involved in the pathological process of myocardial injury and has potential therapeutic value in protecting cardiac function.	Xu et al. (2019) Tong et al. (2017) Gupta et al. (2019) Maeda et al. (2019);Wu et al. (2014) Bian et al. (2020) Deng et al. (2020) Peng et al. (2021) Huang et al. (2020) Li et al. (2019) Wu et al. (2021) Liu (2020) Wan et al. (2019) Xie et al. (2018) Ye et al. (2021)

COPD, chronic obstructive pulmonary disease.

Bone-Related Diseases

Osteoporosis

Cellular senescence, induced by pathways involving p16 or p21, contributes to tissue dysfunction associated with aging and various conditions. However, the specific role of these pathways in senescence related to osteoporosis remains unclear. A study by Khosla et al. utilized 2 parallel genetic models, p21-ATCC and p16-ATCC, to selectively eliminate p21^high and p16^high senescent cells, respectively. The findings indicated that only the elimination of p21^high senescent cells prevented radiation-induced osteoporosis and increased marrow adiposity. This suggests that p21-mediated cellular senescence is a key driver of radiation-induced osteoporosis (Chandra et al., 2022). Therefore, inhibiting osteoblast senescence is a therapeutic method to prevent or delay osteoporosis. Studies have found that the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) in aged rats decreases and the adipogenic capacity increases. Low-magnitude vibration therapy can pass sirt1/p53/p21 axis, eliminate senescent osteoblasts, and promote bone formation in aged rats (Wen et al., 2021). Additionally, mice deficient in cyclophilin B (CypB) exhibit early-onset aging-related phenotypes and markedly elevated levels of p21. Knocking out p21 in these mice alleviates bone defects, indicating that the p21-dependent pathway is partially involved in osteogenesis imperfecta and is closely associated with aging (Zhang et al., 2022).

Osteoarthritis

The role of p21 in the pathology of osteoarthritis is both beneficial and detrimental.

p21-dependent cellular senescence plays a significant role in the pathogenesis of osteoarthritis. Studies that have conducted genome-wide transcriptome analysis on cartilage and synovial tissue from individuals with osteoarthritis and healthy controls have identified p21 as a central gene in arthritis development through differential gene expression and functional enrichment analysis (Shi et al., 2021). Given that aging is a major risk factor for osteoarthritis, CCN3, a novel marker of chondrocyte senescence, promotes this process by inducing p53 and p21, ultimately leading to articular cartilage degeneration (Kuwahara et al., 2020). Sirt1 is also closely related to cellular senescence. It has been shown that the loss of Sirt1 in chondrocytes mediates aging-related secretory phenotypes through the p53/p21 axis, abnormally activating chondrocyte hypertrophy and apoptosis, and accelerating the onset of osteoarthritis (Xu et al., 2020).

Moreover, p21 deficiency can lead to increased susceptibility to osteoarthritis, with p21 knockout mice exhibiting more pronounced osteoarthritis-related phenotypic changes. This suggests that p21 may influence the synovium and cartilage tissue in osteoarthritis by regulating the production of inflammatory cytokines (Takashima et al., 2021).

Metabolic Diseases

The recent discovery of thermogenic beige fat within white adipose tissue presents a promising target for the treatment of obesity and diabetes. Identifying p21 as a key molecular regulator of beige adipocytes offers valuable new insights into potential therapies for metabolic diseases (Park et al., 2021).

However, the premature senescence of AMSCs in white adipose tissue is a major contributor to age-related obesity. Research has found that triglyceride-rich lipoproteins can induce oxidative stress in AMSCs through the SIRT/p53/Ac-p53/p21 axis, leading to an increase in the number of senescent AMSCs, impaired proliferation capacity, and elevated expression of inflammatory factors (Xiang et al., 2020). Additionally, the downregulation of Sprouty has been shown to regulate adipogenesis by inducing p21. Knocking out Sprouty results in the upregulation of p21, causing AMSCs to age and weakening their adipogenic potential. This highlights the specific role of p21, with no involvement of p16 observed, further demonstrating that p21 plays a more critical role in adipogenesis (Mandl et al., 2020).

Insulin resistance, a major risk factor for type 2 diabetes, is frequently linked to obesity. Research by Xu's team using the p21-Cre mouse model has revealed that p21^high senescent cells, primarily located in visceral adipose tissue, contribute significantly to insulin resistance. Transplanting this adipose tissue can induce insulin resistance in recipient mice, whereas the removal of p21^high senescent cells from visceral fat markedly improves insulin resistance. Additionally, treatment with dasatinib and quercetin (D+Q) effectively alleviates symptoms of insulin resistance. These findings underscore the potential of targeting p21^high senescent cells as a therapeutic strategy for metabolic disorders, such as insulin resistance (Wang et al., 2022).

A significant challenge in diabetes management is the development of hyperglycemic memory, where past episodes of high blood sugar continue to affect the body. Research indicates that continuous exposure to elevated blood glucose levels leads to significant alterations in the p21 gene. Studies involving animals, human samples, and in vitro models have shown that high blood sugar persistently induces p21 expression, which is associated with diabetic kidney disease. Targeting p21-dependent renal tubular senescence could, therefore, offer a promising therapeutic strategy to manage hyperglycemic memory. Additionally, elevated p21 levels in the urine of diabetic patients are closely correlated with the severity of diabetic kidney disease, suggesting that p21 could serve both as a mediator and a biomarker for detecting hyperglycemic memory in diabetic nephropathy. By stratifying patients based on urine p21 levels, clinicians can more accurately identify those with advanced renal injury and established renal hyperglycemic memory. For these patients, merely controlling blood sugar may be insufficient; instead, treatments aimed at improving renal tubular senescence should be considered (Al-Dabet et al., 2022).

Moreover, targeting p21^high senescent cells in the endothelium has been shown to prevent vascular dysfunction induced by a high-fat diet (Oh Sung et al., 2023).

Wound Healing

p21-dependent cellular senescence and SASP have been shown to positively impact wound healing. In the following sections, we will explore the role of p21 in various aspects of tissue repair, including epithelial healing, bone tissue damage repair, and the restoration of lung and liver function.

Epithelial Healing

Markers of cellular senescence exhibit varying behaviors in wound healing across different ages. Several studies have compared the expression of aging-related biomarkers in wound healing between young and old subjects, revealing that p21 and p53 play crucial roles in the healing process of younger individuals. This suggests that p21 may be a key factor in human skin wound healing (Chia et al., 2021). Mechanistically, during wound healing, epithelial cells must migrate through the wound to effect repair. Leader cells at the forefront of migrating sheets are activated by p53, which drives their migration. Once activated, p53 regulates p21 activity to guide the differentiation of leader cells while also accelerating the movement of follower cells. This cell migration is a transient process eventually eliminated through cell competition. Research has provided new insights into how p53/p21 drives leader cell behavior and promotes wound healing, offering potential strategies to enhance wound repair (Kozyrska et al., 2022). Additionally, p21 mediates the positive effects of cellular senescence on tissue formation by increasing tissue stiffness and contraction, modifying the expression of extracellular matrix (ECM)-related genes, and facilitating the synthesis of a functional ECM network. This network supports the transfer and storage of cellular forces (Brauer et al., 2023). Recent advancements also include materials that leverage p21's repair functions. For instance, high-concentration trehalose induces a noninflammatory senescence-like state in fibroblasts by upregulating p21, accelerates wound closure, and significantly enhances capillary formation, demonstrating a positive therapeutic effect on wound repair (Muto et al., 2023).

Bone Tissue Damage

p21 has been shown to play a beneficial role in endochondral ossification during fracture healing. p21 deficiency delays ossification by attenuating the Ihh-Runx2-Osterix signaling pathway. In vitro experiments further demonstrate that p21 deficiency in hypertrophic chondrocytes impairs their differentiation (Kikuchi et al., 2022). While p21 does not appear to affect embryonic endochondral osteogenesis, it is crucial for the process of fracture healing.

In contrast, p21 plays a negative role in the repair and healing of articular cartilage. Studies have shown that p21 knockout mice exhibit enhanced healing of articular cartilage damage. The absence of p21 leads to increased chondrocyte proliferation rather than promoting differentiation, highlighting p21's significant role in cartilage repair (Ibaraki et al., 2020). Additionally, p21 knockout mice show a marked increase in cartilage thickness and improved cartilage healing outcomes compared to wild-type mice following full-thickness cartilage defects (Jablonski et al., 2021).

Lung/Liver Damage

The p53/p21 pathway is activated in lung injury as an antiapoptotic mechanism to mitigate damage. However, the deletion of p21 exacerbates lung injury by increasing apoptosis. It is important to note that, while p21 can help alleviate lung injury, its activation may also induce aging as a side effect (Blázquez-Prieto et al., 2021).

In contrast, p21 predominantly acts as a senescence-inducing factor in liver injury. During liver regeneration following partial hepatectomy, p21 mediates senescence in sinusoidal endothelial cells. Notch signaling also induces liver sinusoidal endothelial cell senescence in response to shear stress. However, activating Sirt1 can counteract the upregulation of p21 and other factors, thereby reducing senescence and accelerating regeneration. This suggests that targeting senescent cells could enhance liver repair after injury and offer potential therapeutic opportunities (Duan et al., 2021).

Lung Diseases

Chronic Obstructive Pulmonary Disease

p21-dependent cellular senescence is closely linked to the onset and progression of chronic obstructive pulmonary disease (COPD). Analyzing microarray data from a specific dataset identified senescence-related p21 as a central gene, suggesting that p21 could be a valuable biomarker for the diagnosis and treatment of COPD (Yang et al., 2022). Cellular senescence is a significant risk factor in COPD progression, with bioinformatics analysis revealing that p21 expression levels strongly predict COPD outcomes. Research indicates that the regulatory network involving p21 is closely associated with COPD progression and contributes to the aging process. Thus, p21 holds potential as a novel target for COPD diagnosis and treatment (Zhong et al., 2022). p21 also plays a role in promoting chronic lung inflammation through its regulation of cellular senescence. In p21 knockout mice, the reduction of senescent cells leads to alleviated pathological manifestations of chronic lung inflammation, indicating that p21 is a key regulator of chronic bronchitis and a contributing factor to chronic airway inflammation and lung damage (Levi et al., 2023).

Pulmonary Fibrosis

p21-dependent cellular senescence plays a crucial role in the development of pulmonary fibrosis, characterized by the senescence of lung alveolar epithelial cells. Research shows that p21 expression increases over time in type II alveolar cells (AEC2). Elevated p21 levels in AEC2 not only arrest the cell cycle but also disrupt the P300-β-catenin interaction, inhibiting cell differentiation. Additionally, aged AEC2 cells release profibrotic cytokines that activate myofibroblasts. However, knocking out p21 restores alveolar regeneration, revealing the mechanism through which p21 impedes lung regeneration in pulmonary fibrosis. This finding suggests potential strategies for treating pulmonary fibrosis (Lv et al., 2022). It is important to note that p21- and p16-dependent cellular senescence exhibit different behaviors in pulmonary fibrosis progression. p21^high AECs appear rapidly in the early stages of the disease, while the increase in p21^high AECs is relatively slower over time. This indicates that p21^high senescent AEC2 cells play a critical role in the pathogenesis of pulmonary fibrosis and may serve as targets for developing new treatments aimed at specifically addressing senescent cells or SASP molecules (Yamada et al., 2022).

Nervous-Related Diseases

p21 is associated with various neurological diseases, including Parkinson’s disease and spinal muscular atrophy. In Parkinson’s disease, the senescence of p21-dependent dopaminergic neurons contributes to disease pathology. Research has shown that SATB1 can directly inhibit the expression of the proaging factor p21 in these neurons, and the loss of SATB1 leads to the activation of cellular senescence in dopaminergic neurons (Riessland et al., 2019). Furthermore, in spinal muscular atrophy, p21 plays a crucial role in inducing cell cycle arrest and serves as a mediator of skeletal muscle atrophy (Reedich et al., 2021).

On the contrary, the dual-specificity tyrosine phosphorylation-regulated kinase 1A inhibitor, harmine, can restore redox homeostasis, improve neurodegenerative changes, and alleviate cognitive decline by increasing p21 protein levels. This suggests that harmine holds promise as a therapeutic tool for mitigating the progression of Huntington’s disease through its interaction with p21 (Habib et al., 2022).

Cardiovascular Disease

Cardiac Hypertrophy

p21 plays a critical role in regulating autophagy and is involved in the pathogenesis of cardiac hypertrophy. When stimulated with angiotensin II, both H9c2 cells and primary neonatal rat ventricular myocytes show a significant increase in cell area—by 2.51 and 3.67 times, respectively—along with elevated levels of p21 mRNA and protein expression. Interestingly, inhibiting p21 under angiotensin II stimulation leads to an even more pronounced increase in cell surface area. These findings suggest that elevated p21 expression can suppress angiotensin II-induced cardiac hypertrophy (Tong et al., 2017). Moreover, the administration of oridonin (Xu et al., 2019) and calcitriol (Liu et al., 2020) may offer therapeutic benefits for cardiac hypertrophy by activating p21, likely through its role in autophagy regulation.

Myocardial Fibrosis

Fibroblasts play a crucial role in regulating myocardial fibrosis, as they are the primary cell type contributing to the aging of cardiomyocytes. Previous studies have shown that the senescence of fibroblasts is dependent on the p53/p21 pathway (Gupta et al., 2019). Additionally, it has been observed that reduced expression of miR-26a leads to the upregulation of enhancer of zeste homolog 2 (EZH2) and the downregulation of p21. This combination promotes fibroblast proliferation, contributing to the development of myocardial fibrosis (Zhang et al., 2020). These findings suggest that p21 might regulate myocardial remodeling by activating genes associated with myocardial fibrosis. Enhancing p21 expression in fibrotic myocardial tissue could potentially reduce fibroblast proliferation and slow the progression of myocardial fibrosis.

Arterial Lesions

p21 is a key signaling molecule in replicative senescence in human umbilical vein endothelial cells and aortic endothelial cells, contributing to the formation of atherosclerosis. Vascular smooth muscle senescence, which is associated with p21, may play a significant role in the development of atherosclerosis (Maeda et al., 2019). Studies have shown that LincRNA-p21 is significantly downregulated in animal models of atherosclerosis (Wu et al., 2014). Additionally, research by Bian revealed that suppressing long noncoding RNAs activated by DNA damage leads to cell cycle arrest, senescence, and apoptosis in vascular smooth muscle cells via the p53/p21 pathway (Bian et al., 2020). These findings suggest that p21 can inhibit the proliferation of vascular smooth muscle cells and induce apoptosis in the context of atherosclerosis. Therefore, inhibiting p21 expression in atherosclerosis could potentially slow disease progression. It is important to note that the role of p21 in preventing cardiac hypertrophy differs from its role in atherosclerosis. Thus, caution is warranted when considering the use of p21 agonists for treating cardiac hypertrophy and atherosclerosis, as their effects may vary between these conditions.

p21 plays a significant role in the pathogenesis of coronary artery disease. Research has shown that p21 expression is increased in a vasculitis mouse model of Kawasaki disease, leading to the apoptosis of coronary artery smooth muscle cells. Consequently, p21, along with other factors, may serve as a valuable indicator for predicting the development of coronary artery lesions in Kawasaki disease (Deng et al., 2020).

Myocardial Infarction

The role of p21 in cardiomyocyte proliferation has been extensively studied, primarily highlighting its function as an inhibitor in cardiac regeneration (Peng et al., 2021). However, p21 also plays a beneficial role in myocardial infarction. Acetylation of p21 inhibits cardiomyocyte proliferation and cardiac regeneration, while overexpression of SIRT1 reduces p21 acetylation levels, activating p21 ubiquitination through deacetylation, and thereby alleviating the proliferation block in cardiomyocytes. This indicates that SIRT1-induced p21 deacetylation plays a crucial role in cardiomyocyte proliferation, presenting a potential therapeutic strategy for myocardial infarction (Li et al., 2019). Additionally, Wu et al. (2021) found that deletion of low-density lipoprotein receptor-related protein 6 (LRP6) promotes cardiomyocyte proliferation via the p21 signaling pathway, leading to significant myocardial regeneration after myocardial infarction. Similarly, Liu's (2020) experiment demonstrated that reduced expression of p21 in acute myocardial infarction affects the proliferation, apoptosis, and inflammation levels of rat cardiomyocytes by activating the Wnt/β-catenin signaling pathway. These findings suggest that regulating p21 expression could mitigate the occurrence and progression of myocardial infarction.

Finally, p21 has emerged as a significant protein involved in cardiovascular diseases, though its role remains controversial. It has been found that p21 can alleviate lipopolysaccharide-induced cardiac dysfunction by regulating inflammation and oxidative stress, partly through its regulation of autophagy. This study is the first to demonstrate that p21 can interact with LC3B to promote autophagy, ultimately improving cardiac dysfunction (Huang et al., 2020).

Heart Failure

Wan et al. (2018) discovered that miR-499-5p inhibits doxorubicin-induced mitochondrial fission and apoptosis in cardiomyocytes by targeting p21, effectively preventing doxorubicin-induced cardiotoxicity. Additionally, long noncoding RNA-p21 contributes to doxorubicin-related cardiomyocyte senescence by regulating the Wnt/β-catenin signaling pathway, and silencing this long noncoding RNA can effectively prevent doxorubicin cardiotoxicity (Xie et al., 2018). Moreover, Ye et al. (2021) demonstrated that folic acid administration can decelerate cardiac aging and protect the heart by reducing the expression of p53/p21 and p16. Therefore, suppressing p21 expression in heart failure may potentially enhance heart function.

RESEARCH MODEL OF P21-DEPENDENT CELLULAR SENESCENCE

To evaluate the efficacy of antiaging drugs, researchers commonly use a mouse model in which aging is induced by doxorubicin. In this model, markers of senescent cells, such as p21 and p16, are significantly upregulated in liver and kidney tissues. However, treatment with metformin can reverse these changes in marker expression (Sun et al., 2022). It is important to note that while this traditional model effectively induces an increase in p21^high senescent cells, it is not tissue-specific and cannot accurately target or eliminate these cells. Consequently, it provides only a broad understanding of the role of senescent cells. In the following sections, we will explore research models that specifically target p21-dependent cellular senescence (Table 2).

Table 2.

Research models of p21-dependent cellular senescence

Animal model	Characteristic	Used in research	Advantage	Limitations	References
p21-Cre mouse model	Monitor, sort, image, eliminate, or modulate p21high cells in vivo	Deeply explore the role and potential mechanisms of p21high cells in natural aging, metabolic disorders caused by obesity and other diseases.	Does not interfere with or damage endogenous genes; expanding understanding of the biological complexity and heterogeneity of aging.	Breeding takes a long time, and the breeding methods to obtain the target mouse strain are complex and require multiple genetic identifications.	Oh Sung et al.(2023); Wang et al.(2021)
p21-ATCC mouse model	Selectively kills p21Cip1-expressing senescent cells	Radiation-induced osteoporosis.	Targeting the contribution of p21 to aging and guiding the development of new senolytic drugs targeting p21.	It has a single function and can only eliminate p21Cip1-expressing senescent cells.	Chandra et al. (2022)
p21OE mouse model	A mouse model of p21 overexpression	p21 induces several core properties of cellular senescence in skeletal muscle.	Studying the p21-dependent senescence program from the perspective of overexpression.	p21 expression lacks specificity and requires more precise temporal and spatial overexpression control.	Englund et al. (2023)
p21-3MR mouse model	Tissue-level tracing, real-time monitoring, and targeted removal of p21high cells in vivo	Deeply explore the role of p21high cells in aging-related diseases, which can be combined with ganciclovir targeted elimination.	Powerful function, integrating tracking, control and removal functions into one; no need to breed multiple times with other strains.	There is a lack of more precise interventions that can be used for clinical translation to eliminate p21high cells.	Yi et al.(2023)
p21-GFP zebrafish model	Tracing p21high cells in vivo	Tracking of fluorescent cells in vivo in real-time, testing drug off-target toxicity, and assessment of cellular and phenotypic changes.	Zebrafish share high homology in genes associated with human aging and disease. They can be genetically modified relatively easily.	Research on disease models is limited and needs to be translated into mammalian systems.	Morsli et al.(2023)

p21-Cre Mouse Model

By generating a p21-Cre mouse model, in which the p21 promoter drives inducible Cre recombinase, Wang et al. were able to specifically identify cells with high p21 expression. The p21-Cre mice were then crossed with various floxed mice to monitor, classify, image, eliminate, and regulate p21^high cells in vivo. The study found that p21^high cells emerged following the induction of senescence, and their indirect elimination improved the health of the mice (Wang et al., 2021). Therefore, the p21-Cre mouse model serves as a valuable biological tool for gaining deeper insights into p21^high senescent cells.

p21-ATCC Mouse Model

The p16-ATCC mouse model is designed to target the elimination of p16^high cells, while the p21-ATCC mouse model was developed to study the specificity of p21. In the p21-ATCC model, the p21^Cip1 promoter drives a “suicide” transgene encoding an inducible caspase-8, which selectively eliminates p21^high cells upon induction. A parallel comparison of these 2 mouse models revealed that radiation-induced osteoporosis is primarily driven by p21-dependent cellular senescence, rather than p16. This suggests that p21^high cells are the main contributors to the increased senescent cell load and SASP in bone tissue following radiation (Chandra et al., 2022).

p21OE Mouse Model

The p21OE mouse model, which overexpresses p21, has been used to explore the mechanisms of cellular senescence and the pathological characteristics of skeletal muscle. The findings indicate that p21 can induce senescence in skeletal muscle cells, with p21OE mice displaying specific pathological features in their skeletal muscle. These results suggest that p21 alone is sufficient to initiate the cellular senescence program in skeletal muscle, leading to muscle-related dysfunction (Englund et al., 2023). Moreover, studies using the p21OE mouse model have revealed that p21 not only plays a role in cell cycle arrest but also functions as an immune surveillance “scout” and “timer.” This dual role helps the body establish an internal surveillance mechanism, promoting the growth of senescent cells and ultimately contributing to the maintenance of homeostasis.

p21-3MR Mouse Model

To gain deeper insights into p21, our research group developed the p21-3MR mouse model. This was accomplished by inserting an exogenous 3-peak reporter gene into a specific location within the p21 promoter. This model enables the detection, imaging, and elimination of p21^high cells in vivo. Using the doxorubicin-induced senescence model, we observed that clearing p21^high cells significantly improves multiorgan toxicity in mice. Thus, the p21-3MR mouse model presents a promising tool for studying the role of p21^high cells in cellular senescence (Yi et al., 2023).

p21-GFP Zebrafish

The p21-GFP zebrafish model has been developed due to the high homology between genes associated with human aging and disease. In this model, increased GFP expression under the p21 promoter allows for the visualization of p21^high senescent cells as they express green fluorescent protein following radiation exposure (Morsli et al., 2023). This model is a valuable tool for studying aging, offering significant advantages such as lower costs and higher survival rates compared to mammalian models. As a result, it provides an ideal platform for testing antiaging drugs.

THERAPEUTIC TARGETS FOR P21-DEPENDENT CELLULAR SENESCENCE

Clear Senescent Cells—Senolytic Interventions

This study leveraged bioinformatics analysis to identify compounds targeting the senescent cell antiapoptotic pathways, which are often upregulated in senescent cells (Fig. 3). These identified compounds show potential as senolytic drugs, capable of selectively eliminating senescent cells (Chaib et al., 2022).

Fig. 3.

Therapeutic targets of p21 in cellular senescence.

Notably, the Bcl-2 inhibitor, particularly the antiaging drug ABT-737, has been found to eliminate the abnormally prolonged expression of p21 and enhance liver regeneration. This finding suggests that antiaging drugs, such as ABT-737, could potentially promote organ regeneration (Ritschka et al., 2020). Additionally, targeted integration-based endogenous gene tagging has been successfully implemented in Chinese hamster ovary (CHO) cells. By integrating EGFP-human Bcl-2 into the p21 locus, researchers were able to modify the expression pattern of endogenous p21, revealing an interaction between p21 and Bcl-2 expression. This engineering strategy provides a valuable tool for CHO cell engineering, enabling dynamic control of transgene expression in response to the cell's status (Lee et al., 2020).

Among the widely used inhibitors are those targeting p21, which play a significant role in various physiological and pathological processes. KLF6, for instance, inhibits the proliferation, migration, and growth of pulmonary artery smooth muscle cells (PASMCs) via the p21 pathway, which can accelerate the onset of pulmonary arterial hypertension (PAH) while inhibiting cell growth. The specific p21 inhibitor, UC2288, has been shown to partially reverse the proliferation inhibition caused by KLF7 overexpression (Zeng et al., 2023). Additionally, the p21 inhibitor 17-DMAG has demonstrated the ability to induce cartilage formation both in vivo and in vitro, offering potential new treatments for cartilage damage or osteoarthritis (Bertram et al., 2018). Moreover, the use of p21-targeting siRNA in vivo has been shown to locally and transiently inhibit p21 expression, which improves delayed wound healing in elderly mice (Jiang et al., 2020). Targeting p21, therefore, presents a promising clinical strategy for enhancing wound healing in the elderly. However, current siRNA treatment methods suffer from low efficiency and limitations, underscoring the urgent need for the development of more potent p21 inhibitors. These future inhibitors could play a crucial role in reducing scarring and promoting skin regeneration.

Antioxidants and plant extracts have been shown to play a role in delaying cellular aging through the modulation of p21. For instance, the antioxidant Trolox has been found to reduce the recruitment of FoxO1, diminish the activation of PARIS by FoxO1, and mitigate p21-dependent myoblast cellular senescence caused by oxidative stress (Bae et al., 2020). Similarly, ferulic acid, a compound extracted from plants, promotes the production of nitric oxide in vascular smooth muscle cells via the endothelial nitric oxide synthase pathway. This, in turn, enhances p21 expression and inhibits cell migration and proliferation, thereby contributing to the prevention of atherosclerosis (Wu et al., 2022). Another example is black ginseng, which has been found to influence the Wnt signaling and p53/p21 pathways in metabolic organs and white adipose tissue. By down-regulating the complement system, black ginseng shows promise as a senolytic agent capable of slowing down cellular aging (Lee et al., 2022). Additionally, insulin-like growth factor binding protein 7 has been discovered to activate the biological activity of SIRT1 deacetylase through metabolic processes. This activation leads to the deacetylation of H3K36ac, thereby reducing its binding affinity to the p21 promoter and consequently lowering p21 transcription. As a result, this mechanism can help prevent aging and enhance the regeneration of mesenchymal stem cells derived from dental pulp (Li et al., 2022).

Slowing Down the Aging Process—Senomorphic Drugs

In addition to directly eliminating senescent cells, another strategy to mitigate cellular senescence-related phenotypes or diseases involves inhibiting the expression of the SASP. This can be achieved using SASP inhibitors, also known as senomorphics. These inhibitors target various pathways and molecules, such as the transcription factor nuclear factor NF-κB, the JAK/STAT signal transduction pathway, the serine/threonine protein kinase mTOR, and mitochondrial complex-1-related or 4-related targets. By targeting these pathways, senomorphics can weaken the induction and maintenance of SASP in senescent cells, potentially alleviating related diseases and improving health outcomes (Chaib et al., 2022).

Metformin, a well-known antiaging drug, has demonstrated potential in treating PAH, a condition marked by the proliferation of PASMCs and pulmonary vascular remodeling. Research indicates that NONRATT015587.2 targets p21, thereby promoting PASMC proliferation. However, metformin's ability to upregulate p21 and inhibit PASMC proliferation positions it as a promising treatment for PAH (Sun et al., 2022). Similarly, rapamycin induces the long noncoding RNA GUARDIN, which inhibits p21-dependent aging through the LRP130-PGC1α-FOXO4 signaling axis, contributing to its antiaging effects (Sun et al., 2020). Additionally, BMP9 has been shown to improve postmenopausal osteoporosis by reducing aging gene expression and SASP secretion through the Smad-Stat-p21 axis in vivo, thereby inhibiting osteoblast aging (Xu et al., 2022).

Another significant category of antiaging drugs involves hormone-type medications. Nonylphenol, known for causing mitochondrial dysfunction and oxidative stress, adversely affects pancreatic function. However, melatonin can restore pancreatic function by inhibiting apoptosis through the MDM2-p53-p21 axis (Tao et al., 2022). In conditions such as obesity and other chronic skeletal muscle aging diseases, p21^high cells contribute to mitochondrial dysfunction and increased ROS production. Tamoxifen, on the other hand, can target and eliminate p21^high cells, thereby enhancing muscle performance (Noh et al., 2022).

Currently, extensive research is focused on the combined use of senolytic interventions and senomorphic drugs. This approach integrates 2 pathways: delaying aging and clearing senescent cells. Studies have shown that targeting p21^high senescent cells can yield superior outcomes. For example, the oral administration of dasatinib and quercetin has been effective in eliminating senescent cells, including p21^high cells. The removal of these cells has been particularly effective in reducing renal dysfunction in a renal artery stenosis model (Kim et al., 2021). Additionally, in the p21-Cre mouse model, the senolytic combination of dasatinib and quercetin has been observed to improve insulin resistance symptoms (Wang et al., 2022). Furthermore, in the p21-GFP zebrafish model, a significant decrease in the number of p21:GFP cells was noted after just 5 days of combination treatment with dasatinib and quercetin following irradiation (Morsli et al., 2023). However, despite these promising results, the extreme heterogeneity and dynamic nature of senescent cells in the body present significant challenges to the accuracy and controllability of these treatment methods.

Rejuvenation of Senescent Cells—Senoreventors

The current primary treatment approach for rejuvenating senescent cells involves utilizing stem cells or stem cell-derived extracellular vesicles. For instance, BMSCs are pivotal in regulating immune function. Research has shown that BMSCs can enhance tissue repair in aging spleens and thymuses while significantly inhibiting the production of cytokines such as p21, p16, and IL-10. Additionally, BMSCs can modulate oxidative stress and proliferating cell nuclear antigen levels, ultimately leading to the rejuvenation of aging organs (Wang et al., 2020).

Studies have investigated the infusion of human monocytes engineered to overexpress p21. This overexpression can drive the differentiation of macrophages into tumor-associated macrophages (TAMs) with enhanced phagocytic capabilities. This process has the potential to reduce leukemia burden and extend survival in mice, suggesting that p21 plays a role in directing the proinflammatory reprogramming of TAMs. Consequently, p21 shows promise as a target for immunotherapy (Allouch et al., 2022). Additionally, there is emerging interest in the use of stem cell-derived extracellular vesicles to induce p21-dependent cellular senescence, though this area of research is still in its early stages (Fig. 4; Table 3).

Fig. 4.

Therapeutic targets and signaling pathways for p21-dependent cellular senescence.

Table 3.

Therapeutic targets of p21 in cellular senescence

Senotherapeutic approach	Type	Examples		References
Senolytic interventions	Apoptosis inducers	The Bcl-2 family protein inhibitor	ABT-737, a CHO cell engineering tool targeting Bcl-2	Ritschka et al. (2020); Lee et al. (2020); Sun et al. (2020)
		p21 inhibitors	UC2288, DMAG, p21-siRNA	Sun et al. (2022); Bertram et al. (2018); Jiang et al. (2020)
		Antioxidants	Trolox	Bae et al. (2020)
		Others	Ferulic acid, black ginseng, insulin-like growth factor binding proteins 7	Wu et al. (2022); Lee et al. (2022); Li et al. (2022)
Senomorphic drugs	SASP regulators	Metformin, rapamycin, BMP9, melatonin, tamoxifen		Sun et al. (2022); Sun et al. (2020); Xu et al. (2022); Tao et al. (2022); Noh et al. (2022);
Combined use	Senolytics and senomorphics	Dasatinib and quercetin		Wang et al. (2022); Morsli et al. (2023);
Stem cells and others	Stem cells	Bone marrow MSCs		Wang et al. (2020);
	Targeting the reprogramming and phagocytic capacities of tumor-associated macrophages			Allouch et al. (2022)

CONCLUSION

Senescence-related signaling pathways, including p21 and p16, are well-established markers of aging (Gasek et al., 2021, Gorgoulis et al., 2019). However, accurately interpreting these markers in the context of senescent cells remains a significant challenge due to the inherent heterogeneity of cellular senescence. Most studies have shown that senescent cells are dispersed among various cell populations rather than forming distinct clusters (Cohn et al., 2023). Additionally, the effectiveness of current antiaging drugs varies across different senescent cell populations (Chang et al., 2016, Yousefzadeh et al., 2018, Zhu et al., 2015). Therefore, there is a pressing need to explore more precise approaches that combine appropriate dosing with targeted administration methods to selectively eliminate pathological senescent cells while preserving healthy ones.

At the transcriptional level, p16 and p21 are widely used to identify senescent cells across various tissues, whether due to aging or pathological conditions. However, it is important to recognize that these markers are not universally present in all senescent cells and are not entirely sufficient for detection. The heterogeneity of senescent cells, both in vivo and in vitro, can be influenced by factors such as age, gender, pathological status, tissue location, microenvironment, and the dynamics of cell accumulation (Cohn et al., 2023). Thus, future studies must employ high-resolution techniques to comprehensively characterize the different types of senescent cells under various conditions within the body. Currently, innovative machine learning and artificial intelligence technologies are being developed to distinguish senescent cells from nonsenescent cells based on morphological differences (Suryadevara et al., 2024). Coupled with transgenic model animals and advanced imaging platforms, these advancements are expected to significantly enhance our understanding of cellular senescence in the near future.

Many recent studies have shown that p21^high cells and p16^high cells represent distinct cell populations, differing in cell type, tissue location, accumulation kinetics, and physiological effects (Wang et al., 2022). Specifically, p21^high cells in adipose tissue exhibit several hallmark features of aging, especially in the contexts of aging and obesity (Wang et al., 2021). These findings mark the beginning of a deeper understanding of the diverse roles that various senescent cells play within the body. Therefore, further investigation into the heterogeneity of cellular senescence across different cells and tissues, coupled with the development of more precise methods to selectively eliminate harmful senescent cells at optimal times, is essential. This approach holds the potential to delay aging, rejuvenate tissues, and improve therapeutic outcomes while minimizing the risk of side effects. In conclusion, comprehending the specific roles of different senescent cell types and creating new mouse models are of paramount importance for studying senescence heterogeneity and designing innovative senolytic therapies.

Funding and Support

This work was supported by the Science and Technology Committee Foundation of Shanghai (grant no. 23141902500) and the Natural Science Foundation of Shanghai (grant no. 23ZR1469100).

Author Contributions

Y.J.Y. participated in the initial conceptualization and was a major contributor in writing the manuscript. Z.M.Y. designed and made all figures. R.W.Z., J.Y.Z., and S.W.D. helped with literature search. S.Y.C. and J.H.W. critically revised the manuscript and made vital suggestions in revision. All authors reviewed and approved the final manuscript.

Declaration of Competing Interests

All authors declare that they have no conflict of interest.

Data Availability Statement

Data sharing is not applicable to this article as no datasets were generated or analyzed during the current study.