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Classical and Nonclassical Intercellular Communication in Senescence and Ageing

sasp senescence ageing extracellular vesicles intercellular communication metabolites

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#1 Engadin

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Posted 07 June 2020 - 09:51 PM







S O U R C E :   Trends in Cell Biology







  • Intercellular communication is a key feature in physiological and pathological conditions. We hypothesize that several means of intercellular communication occur either simultaneously or in succession.
  • The most studied means of intercellular communication are soluble factors. However, important alternative means are emerging.
  • Senescent cells are highly proactive and communicate with neighboring cells via various means of intercellular communication including but not limited to the senescence-associated secretory phenotype (SASP).
  • Most studies of pharmacological drugs preventing the release of soluble factors oversee the influence of these drugs on other means of communication.
Intercellular communication refers to the different ways through which cells communicate with each other and transfer a variety of messages. These communication methods involve a number of different processes that occur individually or simultaneously, which change depending on the physiological or pathological context. The best characterized means of intercellular communication is the release of soluble factors that affect the function of neighboring cells. However, there are many other ways by which cells can communicate with each other. Here, we review the different means of intercellular communication including soluble factors in the context of senescence, ageing, and age-related diseases.
Cellular Senescence: A Complex and Heterogeneous Phenotype
The discovery of a cellular phenotype termed senescence (see Glossary) was first identified by Moorehead and Hayflick in the 1960s. When culturing in vitro primary fibroblasts isolated from human donors, they observed that these cells reached a point where they lost their proliferative capacity, and termed this phenotype cellular senescence. Cleverly, they hypothesized that this phenotype could mimic ageing and be exploited as ‘ageing in a Petri dish’ [1,2]. The premature induction of senescence termed oncogene-induced senescence was identified in vitro and later confirmed to play a physiological role in preventing tumor progression in vivo [1,3]. However, it was not until 2011 that the van Deusen laboratory established a causative role between the activation of senescence and ageing [4]. Here, the authors established that the accumulation of p16Ink4a in certain organs in a prematurely aged mouse model deficient for the mitotic checkpoint protein BubR1 triggers natural features common in premature ageing. Interestingly, they showed for the first time that genetic inactivation of p16Ink4a ameliorated these ageing phenotypes [4]. It is now well established that the induction of cellular senescence is a hallmark of ageing [5]. Furthermore, senescence is a driver not only of ageing but also of certain age-related diseases such as cancer, osteoarthritis, atherosclerosis, Alzheimer’s diseases, chronic obstructive pulmonary disease (COPD), and idiopathic pulmonary fibrosis (IPF) among others [6., 7., 8., 9., 10., 11.].
Although the main characteristic of senescence is a stable cell cycle arrest induced by the expression of the cell cycle inhibitors p16INK4A and p21CIP, the influence that senescence has on tissue homeostasis is due to its highly proactive secretome. The senescence-associated secretory phenotype (SASP) could be considered the ‘soul’ of senescence as it is highly proactive and it changes its composition with time. It has both beneficial and detrimental effects depending on the trigger and context where senescence is induced [12,13]. However, the SASP is still not well characterized, as only a number of factors have been identified in very specific scenarios. It is important to note that senescence is a complex heterogeneous cellular phenotype that affects tissue homeostasis in many different contexts and caution should be taken when it is standardized to certain markers (Box 1). It is likely that there remain novel, unveiled characteristics of senescence that are context dependent [14].


Box 1

Guidelines to Identify Senescent Cells In Vitro
Cellular senescence can be induced by a variety of triggers, including telomere shortening, oncogenic stress, ROS, and DNA damage. The main response of primary cells entering senescence is to induce a stable cell cycle arrest by expressing the cell cycle inhibitors CDKN2A, CDKN2B, and/or CDKN1A (encoding p16INK4A, p15INK4B, and/or p21CIP proteins, respectively) and showing a lack of proliferation-related markers such as Ki67, BrdU, or EdU (Figure I) [13,14]. However, as these markers are not exclusive to senescent cells but are also present in nondividing somatic cells, additional markers should be used to confirm a senescent phenotype [89]. The identification of more than three biomarkers is recommended to confirm the activation of senescence. Another marker used to identify senescence is senescence-associated beta galactosidase (SA-β-Gal) activity. An increase in SA-β-Gal is due to higher lysosomal activity in senescent cells, which could be due to an increase in the number of lysosomes in senescence [37] or an increase in lysosomal activity and can be detected by a specific stain. Finally, additional markers such as DNA damage, the release of a particular secretome that has been termed the SASP [12], or the activation of specific signalling pathways should also be verified. However, these last markers are slightly more challenging as some triggers, such as oncogene-induced senescence by H-RasG12V expression, induce all of them simultaneously. Instead, other triggers, such as developmental senescence or an increase in αvβ3, do not induce a DNA damage response, which was long believed to be a key biomarker of senescence [65,73,74]. This confirms that not all biomarkers are expressed simultaneously when senescence is induced, adding intricacy to the identification of senescence.
It is important to note that senescence is an extremely complex and heterogeneous cellular phenotype that affects tissue homeostasis in many different contexts, and the generalization of standardized markers might be disadvantageous as it is likely that there are as-yet-unveiled characteristics of senescence that are context dependent [14].




Figure I. Biomarkers of Senescence.
Abbreviations: EV, extracellular vesicle; mTOR, mammalian target of rapamycin; SA-β-Gal, senescence-associated beta galactosidase; SASP, senescence-associated secretory phenotype; TGF-β, transforming growth factor β.
There are currently two main therapeutic strategies to deal with the presence of senescent cells in ageing and age-related diseases. One relates to the potential for selective killing of senescent cells (using pharmacological compounds termed senolytics). The second aims to neutralize the deleterious effects of intercellular communication, in particular the SASP, on senescent cells by using drugs denominated senomorphics. Here, we review the different means of intercellular communication for senescent cells and their relation to ageing and some age-related diseases, classifying them as classical, emerging, and nonclassical. We include soluble factors and extracellular matrix (ECM) remodeling proteins, but also describe additional ways by which cells communicate. We focus on how intercellular communication is regulated and its functionality in different biological and age-related pathological contexts.
SASP: Classical and Nonclassical Intercellular Communication
The SASP is a means of intercellular communication that specifically refers to senescent cells. The classical SASP is characterized by soluble factors, growth factors, and ECM remodeling enzymes [12]. However, emerging SASP and other means of intercellular communication that we have denominated nonclassical have also been described during senescence and ageing. Here, we review the current knowledge on what we call classical, emerging, and nonclassical SASP.
Classical SASP: Soluble Factors or sSASP
The sSASP can be both beneficial and detrimental for tissue homeostasis; therefore, the sSASP needs to be tightly regulated. One of the main drivers of the sSASP is a persistent DNA damage response [15]. This converges in the activation of two major regulators of the SASP: NF-κB and C/EBPβ, where NF-κB is further regulated by the transcription factor GATA4 (Figure 1A) [16]. However, the sSASP can also be induced independent of a noncanonical DNA damage response by p38MAPK [17] and by the presence of cytoplasmic chromatin fragments (CCFs). These are DNA fragments that can be released from the nucleus during senescence and activate the antiviral cyclic GMP-AMP synthase (cGAS) stimulator of interferon genes (STING) pathway [18,19]. Still, both p38MAPK and CCF activate the sSASP via NF-κB signaling.
Figure 1. Intercellular Communication in Senescence and Ageing.
(A) Soluble factors, growth factors, and matrix remodeling enzymes are released in the classical soluble senescence-associated secretory phenotype (sSASP) model. However, the sSASP needs to be tightly regulated. At the transcriptional level, BRD4 and GATA4 regulate the master regulators of the SASP: NF-κB and C/EBPβ. The sSASP can also be driven by LINE-1 (L1) retrotransposable elements that are derepressed during senescence and by cytoplasmic chromatin fragments (CCFs) through the activation of cyclic GMP-AMP synthase (cGAS). The sSASP is also regulated post-transcriptionally by mammalian target of rapamycin (mTOR) and downstream signalling pathways. The secreted sSASP plays many roles in the microenvironment. On the one hand, it alters the extracellular matrix (ECM) and can reinforce senescence through autocrine signalling. On the other hand, it has many functions through paracrine signalling. It can promote cancer progression, stem cell self-renewal, and stem cell-like features in damaged cells. It also has the ability to transmit senescence to neighboring cells. Importantly, the sSASP stimulates an innate and adaptive immune response favoring the elimination of senescent cells. (B) Emerging SASP factors include extracellular vesicles (EVs) (evSASP). Due to the high heterogeneity and cargo diversity of EVs released during senescence, novel functions are continually emerging. Some cargo factors, such as interferon (IFN) and ephrin-related and antioxidant proteins, are highlighted. In addition, miRNAs (miRs) and CCF DNA fragments can be loaded into evSASPs. evSASPs also exert paracrine functions to promote cancer progression and induce paracrine senescence. Furthermore, metabolites and lipid mediators that induce an oxidative microenvironment or favor tumor progression have been found. Gut microbiota release lipoteichoic acid (LTA) and deoxycholic acid (DCA), which drive a sSASP response via COX-2 and prostaglandin E2 (PGE2) release. © Nonclassical SASP refers to intercellular communication in senescence not driven by released factors. Cell-to-cell communication can be mediated via receptor interaction as with interleukin-1 receptor (IL-1R) and interleukin-1 alpha (IL-1A) or via NOTCH1/JAG interaction, both of which induce sSASP. In addition, cell–ECM interaction has been described in senescence. For example, integrin α6β1 can interact with CCN1 (cysteine-rich protein 1) and induces senescence via reactive oxygen species (ROS) while αvβ3 induces sSASP. Alternatively, cytoplasmic extensions called cytoplasmic bridges also occur during senescence. Interestingly, these have been described between senescent cells and natural killer (NK) cells to activate these and promote the elimination of senescent cells. The fusion of cells can also induce senescence allowing material exchange between the fused cells. Furthermore, senescent cells can engulf cancer cells as a source of energy to maintain the high metabolic requirements of senescent cells. Abbreviations: eNAMPT, extracellular nicotinamide phosphoribosyltransferase; EphA2, ephrin A2; IFITM3, interferon-induced transmembrane protein 3; LTBP, latent TGF-binding protein.
Curiously, activation of the inflammasome can also control the sSASP. This is mediated by interleukin (IL)-1 signaling and IL-1α expression and is mainly involved in paracrine senescence signaling [20]. Furthermore, IL-1A is regulated by mammalian target of rapamycin (mTOR) inhibition. mTOR selectively regulates a number sSASP factors post-transcriptionally via IL-1A, but also through MK2 [mitogen-activated protein kinase-activated protein kinase 2 (MAPKAPK2)] by the protein synthesis factor 4EBP1 [21,22].
Alternatively, epigenetic alterations are also known to regulate the sSASP. A recent study found that derepression of the retrotransposable element line 1 (L1) increased during senescence and ageing. This in turn activated cGAS/STING and, consequently, the sSASP [23]. Furthermore, the sSASP is regulated by global chromatin remodeling and the recruitment of BRD4 to superenhancers near sSASP genes [24]. Overall, the sSASP is tightly regulated as dysfunctional expression and release can drive pathological conditions. This highlights the importance of understanding how the sSASP is regulated.
It was long believed that the sSASP would change in composition throughout time. Thus, it was not until recently that a comprehensive study found the sSASP to comprise two distinctive functional waves [25]. The first wave is formed by an anti-inflammatory transforming growth factor (TGF)-β-enriched sSASP. This particular sSASP is regulated by membrane-bound NOTCH1, whose expression increases during the initial stages of the induction of senescence and mediates C/EBPβ repression. Second and through time, NOTCH1 expression decreases causing the activation of C/EBPβ, which in turn induces a proinflammatory sSASP [25].
Our advancement of knowledge of the sSASP has greatly increased, with many novel roles emerging in the past decades. On the one hand, the sSASP is important for reinforcing a stable cell cycle arrest in an autocrine manner through IL-8 and IL-6 and their corresponding receptors (Figure 1A) [26,27]. On the other hand, it acts in a paracrine fashion influencing a variety of cell types. It can induce senescence in primary fibroblasts and epithelial cells [20], while promoting tumorigenesis in cancer cells [13,14,28]. Importantly, the sSASP also acts in a paracrine fashion by mediating both an innate and an adaptive tissue response favoring the removal of senescent cells from the tissue, which is essential for the maintenance of tissue homeostasis [20,29,30]. More recently, the SASP was shown to be key for tissue regeneration via IL-6 [31,32] and cell plasticity and stemness [33,34]. Despite many recent advances in novel roles and regulation of the sSASP, there are also emerging functions as described next.
Emerging Classical SASPs
Although the SASP is classically defined as the secretome released from senescent cells, most of the current literature focuses on soluble factors, growth factors, and ECM remodeling enzymes. In this section, we aim to review emerging literature on novel SASP components such as extracellular vesicles (EVs) and noncellular metabolites and ions (Figure 1B).
EVs are lipid membrane vesicles that are released by all cells and are therefore found in most biological fluids [35]. Although initially thought of as a mechanism to release unwanted components from the cell, they are now recognized as a well-established mechanism for intercellular communication. Despite this, senescent cells remove toxic cytoplasmic DNA via EVs to maintain cellular homeostasis [36]. Indeed, senescent cells release more EVs (evSASP) than proliferating cells [36., 37., 38., 39., 40.]. Interestingly, it has been shown that these EVs induce paracrine senescence in healthy cells, highlighting their importance as intercellular communication mediators (Figure 1B) [37,39]. While the mechanisms responsible for this are unknown, several miRNAs, interferon-related proteins, and antiapoptotic proteins were found enriched in senescent-derived EVs [37,39., 40., 41., 42.]. By contrast, a protumorigenic role for EVs derived from senescent cells mediated by ephrin A2 (EphA2) has been suggested [38] adding an extra layer of complexity to the evSASP. Interestingly, several ephrin-related proteins are enriched in plasma derived from old healthy donors [43,44], although whether they are free proteins or EV associated remains to be determined.
Metabolites are small chemical byproduct molecules of metabolic activity in cells and tissues that provide functional evidence of biochemical activity [45]. Thus, several metabolites are emerging as biomarkers of senescence, ageing, and related diseases. Interestingly, metabolite regulators have also been found inside EVs conferring intrinsic metabolic activity on EVs. For example, extracellular nicotinamide phosphoribosyltransferase (eNAMPT), a regulator of NAD+, was found in EVs and decreased with ageing in mice and humans. Furthermore, EV-contained eNAMPT increased NAD+ levels in recipient cells, delaying ageing and extending lifespan in mice [46]. Interestingly, NAMPT has been shown to regulate the SASP [47].
The extracellular metabolite profile differs between young and elderly individuals. Analysis of blood from old donors (>74 years old) showed a reduction in metabolites related to antioxidants, redox, and muscle maintenance [48]. Levels of the antioxidant molecule NAD+ decrease in various tissues, including plasma [49], during ageing [50,51], which correlates with the reduction in the relative NAD+:NADH ratios found in mitochondrial dysfunction-associated senescence [52]. Together these findings are associated with the extracellular metabolic profile of senescent cells, where an increase in citrate and metabolites involved in oxidative stress were found [53]. Citrate, however, can be found associated with iron in the blood. The levels of iron are generally increased in ageing and age-related diseases as Alzheimer’s and Parkinson’s, leading to the generation of reactive oxygen species (ROS). ROS, lactate, ketones, glutamine, and nitric oxide (NO) are all high-energy metabolites released by senescent cells producing a toxic surrounding microenvironment [54]. Given that ions and metabolites have been poorly characterized in senescence and ageing, it will be critical to further understand their implications as emerging components of the SASP.
Lipid mediators involve a family of molecules implicated in anti- and proinflammatory mechanisms that also enhance microbial clearance [55]. A recent study found that the obesity-related gut microbiota components lipoteichoic acid (LTA) and deoxycholic acid (DCA) induce senescence in hepatic stellate cells (HSCs), promoting hepatocellular carcinoma (HCC) progression [56,57]. Interestingly, LTA induced the release of the lipid metabolite prostaglandin E2 (PGE2) via COX-2 and suppressed the antitumor immunity. It would be interesting to determine whether other lipid mediators are implicated in tissue homeostasis maintenance as part of the emerging SASP.
Nonclassical Intercellular Communication
In this section, we review other means of intercellular communication beyond soluble and emerging factors that are also important during senescence and ageing. We call these nonclassical (or not secreted) mechanisms for intercellular communication (Figure 1C).
Juxtacrine signaling is a means of intercellular communication characterized by cells involving ligand–receptor binding. Senescent cells also communicate with their neighbor cells through cell-to-cell or juxtacrine contact. For example, IL-1A, thought to be a master regulator of soluble factor paracrine senescence [20], also regulates juxtacrine senescence [58]. Senescent cells express membrane-bound IL-1A, which interacts with the IL-1R to control the levels of IL-6 and IL-8; thus, downregulation of IL-1R or IL-1α using RNAi or blocking antibodies prevents the upregulation of IL-6 and IL-8 during senescence (Figure 1C) [58]. Furthermore, in an elegant study Hoare et al. showed that membrane-bound NOTCH1 expression regulates the composition of the SASP, which they found to be highly dynamic [25]. Although NOTCH1 initially drives a classical TGF-β-enriched secretome, it also contributes to senescence mediated by cell-to-cell contact through the juxtacrine NOTCH/JAG pathway in a lateral induction fashion [59]. This has been defined as secondary juxtacrine senescence, where NOTCH1 is essential for the transmission of senescence through cell-to cell signaling but not for paracrine senescence mediated by soluble factors or cell-autonomous senescence [60].
It is known that sSASP can induce ECM remodeling and stiffening, which can alter immune cell recruitment in ageing [54]. Although it is acknowledged that senescent cells secrete a variety of ECM remodeling proteins, the interaction of the senescent cell with the ECM is less well described. Senescence can be induced by the interaction between the integrin α6β1 and the matricellular protein CCN1 during wound healing, inflammation, fibrosis, heart regeneration, and cancer, activating ROS [61., 62., 63., 64.]. Furthermore, integrin αvβ3 was also shown to induce senescence by activation of the TGF-β pathway in a cell-autonomous and non-cell-autonomous fashion [65,66]. However, although ROS release was induced, there was no DNA damage associated, suggesting that the ECM–cell interaction can induce senescence via different mechanisms.
Cell-to-cell fusion is a means of intercellular communication that can be induced upon the aberrant expression of fusogenic proteins or in response to viral infection such as with measles or ERVWE1 expression [67]. It induces senescence in response to fusion and mediates an immune response not only in primary but also in cancer cells. Although the mechanisms implicated remain to be elucidated, p53 is partially needed for the induction of senescence by fusion in cancer cells [68]. One of the pathological conditions where cell fusion has been observed is cancer [67]. Interestingly, the activation of senescence by chemotherapy in cancer cells stimulates them to ‘engulf’ neighboring cells, which are later processed through the lysosome. This provides these cells with material and energy to sustain the required high metabolic capacity of senescent cells [69]. However, the mechanisms behind this and whether this is a form of cell fusion would need to be further addressed.
Besides cell fusion, cytoplasmic bridges also allow the exchange of biological material between cells, including RNA, proteins, and even organelles such as mitochondria and lysosomes [70,71]. Cytoplasmic bridges are membrane extensions that allow spatiotemporal interaction between nearby cells. The transfer of materials between cells has been proved in diverse cell types, such as neurons, cancer cells, and immune cells, but also senescent cells [70,71]. By performing trans-stable-isotope labelling of amino acids in cell culture (SILAC), Biran et al. showed that senescent cells transfer protein material to natural killer (NK) cells through cytoplasmic bridges mediating their activation and increased cytotoxicity [70]. The transfer is dependent on the GTPase CDC42 and the proteins transferred are mainly involved in actin cytoskeleton reorganization and antigen presentation, although certain proteins important for the activation of NK cells such as HSPA5 and CALR were also found. Interestingly, protein transfer also happens in vivo [70], clear whether the transfer is mediated exclusively by cytoplasmic bridges or by additional intercellular communication mechanisms.
Intercellular Communication in Physiology and Pathology
Together, the described studies show the importance of the contribution of classical, emerging, and nonclassical intercellular communication occurring during senescence and ageing. Although we have categorized the means for intercellular communication in the different sections, the reality is that it is extremely likely that a simultaneous combination of some, if not all, is what occurs in vivo, which will further depend on the biological or pathological context. Here, we review which means of communication are known for senescence in a particular physiological or pathological scenario. In addition, we discuss described communication types where we can hypothesize a role for senescence in particular contexts.
Intercellular Communication Contributes to Tissue Homeostasis Maintenance
The development of the placenta is an important step during pregnancy. One of the outer layers of the placenta is the syncytiotrophoblast that is formed by the fusion of trophoblast cells. This fusion induces the activation of senescence in vitro and several markers of senescence have been observed in this structure in vivo [68]. Although it is unclear why cells in the syncytiotrophoblast become senescent, it is speculated that it could be a mechanism to prevent apoptosis or contribute to maternal–embryonic transport in the placenta [68]. EVs from trophoblast cells have been shown to deliver miR-enriched EVs to non-trophoblast cells to protect against viral infections by inducing autophagy [72]. Together with the ability of senescent cells to develop cytoplasmic bridges, these mechanisms seem to potentiate the exchange of materials between cells during development (Figure 2A, top panel) [70,72].
Figure 2. Intercellular Communication in Physiology and Age-Related Pathology.
(A,B) Different types of intercellular communication have been described that can be (A) beneficial or (B) detrimental for tissue homeostasis and organism wellbeing. Blue boxes indicate that there is scientific evidence for a correlation between that type of intercellular communication and senescence; orange boxes show scientific evidence for the indicated particular physiological or pathological situation but not in the context of senescence. Abbreviations: ECM, extracellular matrix; evSASP, extracellular vesicle senescence-associated secretory phenotype; sSASP, soluble senescence-associated secretory phenotype.
Senescence is a normal process during development, where the sSASP plays a key role in mediating the clearance of these cells by macrophages to ensure the correct formation of the embryo [73,74]. The sSASP induces tissue remodeling and regeneration, a process in which cell fusion plays a critical role [75]. Interestingly, TGF-β as part of the sSASP seems to be key for senescence during development [73]. As integrins and NOTCH signaling regulate TGF-β in senescence [25,65] and both play key roles in development [76,77], it would be interesting to understand whether juxtacrine and ECM–cell intercellular communication occur in senescent cells at this time.


Also tagged with one or more of these keywords: sasp, senescence, ageing, extracellular vesicles, intercellular communication, metabolites

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