Cellular senescence in vivo: From cells to tissues to pathologies
Introduction
Cellular senescence is a form of stable cell cycle arrest that occurs in diploid cells when they are exposed to certain stressors, including genotoxic agents, nutrient deprivation, hypoxia, mitochondrial dysfunction and oncogene activation. Senescence was first described in primary cells by Hayflick and colleagues (Hayflick and Moorhead, 1961), when they observed that human diploid fibroblasts in vitro could reach only a finite number of cell divisions before their proliferation was arrested irreversibly. This biological phenomenon, referred to as the “Hayflick limit”, was later attributed to progressive telomere shortening upon consecutive rounds of cell divisions (Courtois-Cox et al., 2008). The Hayflick limit represents a physiological cellular response to counter genomic instability (Courtois-Cox et al., 2008; Hayflick and Moorhead, 1961). This phenomenon is now also referred to as replicative senescence. However, an accelerated senescence response, which is independent of telomere shortening, can also arrest growth or proliferation of diploid cells, and it is known as premature senescence (Courtois-Cox et al., 2008; d’Adda di Fagagna et al., 2003; Serrano et al., 1997). Subsequently, it was demonstrated that oncogenic stress (e.g., overexpression of oncogenes and loss of tumor suppressor genes) can induce premature senescence in vitro, a process known as ‘oncogene-induced senescence’ (Counter et al., 1992; Serrano et al., 1997).
Many questions regarding the causes, signaling networks and mechanisms underlying the various types of cellular senescence still remain open, and current evidence is largely based on in vitro experiments. At the molecular level, cell cycle arrest and exit is controlled by the activation of the p53/p21CIP1 and the p16INK4a/Rb tumor suppressor pathways (Herranz and Gil, 2018). The senescence growth arrest is often triggered by a persistent DNA damage response (DDR) caused by either intrinsic (e.g. oxidative damage, telomere attrition, hyperproliferation) or extrinsic insults (e.g. ultraviolet- or γ-irradiation, cytostatic drugs) (Herranz and Gil, 2018). In the context of the DDR signaling pathway, ATM or ATR kinases block cell-cycle progression through stabilization of p53 and transcriptional activation of the cyclin-dependent kinase (Cdk) inhibitor p21 (van Deursen, 2014). Moreover, activated oncogenes, such as oncogenic Ras, are also prominent inducers of senescence. Oncogenic Ras acts through overexpression of Cdc6 and suppression of nucleotide metabolism, resulting in aberrant DNA replication, formation of double-strand DNA breaks (DSBs) and activation of the DDR pathway (Aird et al., 2013; Di Micco et al., 2006). However, senescence caused by E2F3 activation or c-Myc inhibition is DDR-independent and involves p19Arf and p16Ink4a (Lazzerini Denchi et al., 2005; Nardella et al., 2011). Senescence is closely linked to profound metabolic changes (Dörr et al., 2013; Kondoh et al., 2005). Furthermore, various tumor suppressors trigger a senescent growth arrest when inactivated, including RB, PTEN, NF1 and VHL (Nardella et al., 2011; Shamma et al., 2009). While RB inactivation involves the DDR (Shamma et al., 2009), the other tumor suppressors mentioned are DDR-independent and induce senescence through p19Arf and p16Ink4a. An interesting species-specific difference is that senescence pathways in murine cells are more dependent on p19Arf than those in human cells (Ben-Porath and Weinberg, 2005). Recently, the cGAS–cGAMP–STING pathway, in the context of which cGAS senses cytoplasmic DNA as a consequence of nuclear DNA damage, has been shown to connect DNA damage to inflammation, senescence and cancer via activation of the transcription factors IRF3 and NF-κB (Li and Chen, 2018; Yang et al., 2017). In summary, many senescence-inducing stressors or stimuli have in common that they trigger a DDR which then orchestrates downstream signaling mediated by ATM/R kinases or the cGAS–STING axis to acquire a full senescence phenotype. On the other hand, some of the stimuli induce cellular senescence through p19Arf, p16Ink4a and profound metabolic changes suggesting that type, intensity and/or exposure time to senescence-inducing stimuli can play a major role in determining which senescence pathway gets triggered. However, it is important to note that current models regarding the molecular mechanisms involved in cellular senescence are largely based on in vitro data. To what extent each of these pathways is involved in driving cellular senescence within different tissues and cell types in vivo is an important issue that remains to be explored in future studies.
A primary feature of senescence cells, which distinguishes them from the state of quiescence, is irreversible proliferative withdrawal. In vitro, senescent cells often exhibit increased size with a flattened morphology, smooth shape, large vacuoles and possibly multiple nuclei. However, these changes in size and shape of senescent cells may not necessarily occur in the same manner in tissues in vivo (Rhinn et al., 2019). An additional feature of senescent cells is that they fail to respond to growth- and apoptosis-inducing stimuli (Baar et al., 2017; Yosef et al., 2016; Zeng et al., 2018). A typical senescence phenotype is characterized by a number of intrinsic and extrinsic markers, although none of them is specific or universal for senescence. At the molecular level, senescent cells are routinely characterized by upregulation of cell-cycle inhibitors such as p21 and/or p16 (Caliò et al., 2015; He et al., 2017), positive staining of senescence-associated β-galactosidase (SA-β-gal) at pH 6 (Dimri et al., 1995; He et al., 2017; Hernandez-Segura et al., 2018), formation of senescence-associated heterochromatin foci (SAHF) (Yamauchi et al., 2017), accumulation of lipofuscin, loss of lamin B1 (Shimi et al., 2011), senescence-associated distension of satellites (Swanson et al., 2013), the induction of senescence-associated DNA damage (Kim et al., 2017) and the secretion of a large number of factors, including growth factors, cytokines, chemokines, and proteases, which is commonly known as the senescence-associated secretory phenotype (SASP) (Coppe et al., 2010; Lopes-Paciencia et al., 2019). Moreover, enhanced expression of p19ARF, p53 and PAI-1 are also observed in senescent cells and are used as senescence markers (Bernardes de Jesus and Blasco, 2012; Hernandez-Segura et al., 2018). A recent study suggested that c-Met could serve as an early marker of cellular senescence; however, the observations are based on in vitro investigation (Boichuck et al., 2019). Recently, Gal et al. demonstrated the use of the ImageStreamX approach as a powerful method for the detection and quantification of senescent cells in distinct tissues and cell populations (Gal et al., 2019). This novel method combines several senescence-related markers, together with the commonly used senescence-associated β-galactosidase assay and thereby offers a new solution to quantify senescent cells in vivo. For a summary of commonly used markers of cellular senescence, see Table 1.
Cellular senescence is induced under physiological or pathological conditions by a variety of extrinsic or intrinsic cellular signals (Avelar et al., 2020b; He and Sharpless, 2017; Hernandez-Segura et al., 2018; Yanai and Fraifeld, 2018; Zeng et al., 2018). The persistence of senescent cells in tissues may have beneficial function as well as may alter tissue remodeling and homeostasis: transient accumulation of senescent cells in tissues is mainly associated with beneficial functions (Calcinotto et al., 2019), whereas long-term accumulation of senescent cells appears to deteriorate tissue homeostasis. Recently, transgenic mouse models have been developed to permit the elimination of senescent cells in vivo in mice (including the INK/ATTAC model, the p16−3MR model and the ARF-DTR model (Baker et al., 2011; Demaria et al., 2014; Hashimoto et al., 2016), and a series of individual studies using these mouse models have now established clear, causal contributions of senescent cells to lifespan, wound healing, tissue development and programming and to age-related functional decline in specific organ systems (Avelar et al., 2020a; Baker et al., 2016, 2011; Childs et al., 2015; Docherty et al., 2019; Farr and Khosla, 2019; Hashimoto et al., 2016; Tacutu et al., 2011). In additional studies, senolytics, drugs that specifically target senescent cells by inducing apoptosis of senescent cells, were also used for the clearance of senescent cells in mice and humans as an alternative approach to the INK/ATTAC model, and interestingly, comparable therapeutic benefits have been reported (Hickson et al., 2019; Short et al., 2019; Zhang et al., 2019). The accumulation of senescent cells with advancing age in various tissues and their role in age-related tissue alterations and pathologies, such as osteoarthritis (Price et al., 2002) and atherosclerosis (Campisi, 2005), has been documented by a number of studies (Calcinotto et al., 2019; He and Sharpless, 2017; Rhinn et al., 2019). It has also been reported that senescent cells accumulate in various tumors in vivo, where they are thought to influence tumor progression (Campisi, 2005; Lee and Schmitt, 2019; Zeng et al., 2018). Therefore, therapeutic approaches which can selectively clear senescent cells may be useful for the treatment or prevention of a broad range of age-related disorders.
Available evidence indicates that only specific cell types within a given tissue are susceptible to acquiring a senescent phenotype, whereas other cell types remain unaffected by senescence. To further study the cells that are being targeted or should be targeted by senolytic approaches, knowledge of their identities is required. Here, we review currently available evidence linking specific cell types in various tissues to the potential for acquiring a state of senescence in vivo (see main text below and Table 2). We have focused this review on tissue types where cellular senescence has been suggested to play a role in functional alterations of a given tissue in vivo. We have not included tissues where no clear evidence for in vivo senescence, based on multiple methods, is available. We also required the availability of data on cell type identity of senescent cells and demonstrated links to tissue function. The data available may serve as an important starting point for the further definition of molecular and functional characteristics of senescent cells in different organs and may hence promote the development and refinement of targeting strategies aimed at removing senescent cells from aging tissues.
Section snippets
Tissue repair and wound healing
Senescent cells have been demonstrated to play a role in wound repair and the fibrotic response in a number of tissues, including the liver (Krizhanovsky et al., 2008), skin (Jun and Lau, 2010), lung (Schafer et al., 2017) and heart (Zhu et al., 2013). In a preclinical mouse model of liver damage and fibrosis, senescent hepatic stellate cells were identified in the fibrotic lesions in liver (Krizhanovsky et al., 2008). In this study, induction of damage in mice deficient for both the p53 and p16
Concluding remarks
There is plenty of evidence suggesting that a variety of cellular stressors and various insults are key contributors to in vivo cellular senescence, which in turn is thought to contribute to age-related tissue dysfunctions and pathologies. Even a relatively small percentage of senescent cells in organs may impair tissue homeostasis and regeneration, decrease organ function, and contribute to aging phenotypes, as demonstrated by those studies where senescent cells were either genetically or
Acknowledgements
This work was supported by a grant from the Helmholtz Future Topic AMPro (Aging and Metabolic Programming).
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