The reactive oxygen species (ROS) is considered as one of the main activators of senescence cells[18,19]. The imbalanced ROS may cause oxidative stress and DNA damage, leading to the activation of DNA damage response in the affected cells.
Signaling pathways that control cell cycle arrest
The p53 and Rb signaling pathways may become activated in response to extrinsic and intrinsic stressors, including DNA damage. p53 and Rb can affect tran-scriptionally both upstream regulators and downstream effectors in the senescence pathway. The inactivation of either p53 or Rb prevents or significantly delays senescence (Figure 1). ATM and ATR, as well as their downstream targets, Chk1 and Chk2, become activated during DNA damage, which in turn activate p53. Activated p53 is considered as one of the main players in converting normal cells to senescent cells. The phosphorylated form of p53 is the essential contributor of replicative senescence in human fibroblast. Acetylation of p53 at Lys161/Lys162 is also important for cell cycle arrest and senescence. Once p53 is activated by posttranslational modification, it in turn activates its transcriptional target, p21Cip1 (CDKN1A). p21Cip1 is the inhibitor of Cyclin E/CDK2 complex and it promotes cell cycle arrest at G1/S phase of cell cycle (Figure 1). Of note, p21Cip1 can be activated by both p53-dependent and p53-independent mechanisms.
Figure 1 Stress-induced senescence.
Both external and internal stresses can induce DNA damage and the activation of p16INK4A and/or p15INK4B. DNA damage can activate p53 ataxia telangiectasia mutated (ATM) and ATM and RAD3-related (ATR) pathway. Activated p53 induces p21 Cip1 expression. Expression of p21Cip1 can also be regulated by p53-independent mechanisms. The Cyclin dependent kinases activate Rb but are inhibited by p15INK4B/ p16INK4A and p21Cip1 which leads to cell cycle arrest and senescence. The senescent cells express senescence-associated secretory phenotype (SASP). The SASP factors may induce senescence in neighboring cells in a paracrine fashion. ATM: Ataxia telangiectasia mutated; SASP: Senescence-associated secretory phenotype.
Retinoblastoma protein (Rb) is a tumor suppressor and a well-established cell cycle regulator. Rb is expressed in all tissues and controls cell cycle progression through interactions with the E2F family of transcription factors[20,25]. Other Rb family members, namely, p107 and p130, also play active and important role in driving senescence independently of Rb. p21Cip1 has been found to be both a positive and negative regulator of Rb by regulating either Rb phosphorylation or degradation, respectively. p16INK4A (CDKN2A) inactivates Cyclin-dependent kinases, which phosphorylate Rb, and Rb phosphorylation status in turn has an impact on the expression of p16INK4A. In normal cells, Cyclin D and Cyclin E bind to CDK2 and CDK4/6, respectively, since p21Cip1 and p16INK4A level remain at the basal level. Hyperphosphorylation of Rb permits the E2F factor to participate in the production of replication proteins and hence cell cycle progress. But a higher level of either p16INK4A and/or p21Cip1 can keep Rb in the hypophosphorylated state so that E2F remains bound with it. Therefore, cell cycle is arrested, which ultimately lead to cellular senescence (Figure 1).
Another cell cycle inhibitor, CDKN3 is a phosphatase and dephosphorylates Rb thus ensuring that cell cycle progression is blocked. Thus, CDKN3 is considered as an important contributor to cellular senescence. CDKN3 can also interact with Mdm2 and form a complex with p53 and Mdm2. Once the complex is formed, p53 losses its ability to induce p21Cip1[29,30].
G1-phase cell cycle arrest can also be induced by transforming growth factor-beta (TGFβ). TGFβ can keep Rb in hypophosphorylated state, suggesting that it may suppress Rb phosphorylation and thereby interfering with cell cycle progression. p15INK4B(CDKN2B), a homologue of p16INK4A, is activated by TGFβ, acts on CyclinD-CDK4/CDK6 complex and contributes to cell cycle arrest, and hence to senescence (Figure 1). Since p15 INK4B and p16INK4A have a common mode of action, in a tissue where either one does not express, the other can possibly compensate the func-tionality.
Signaling pathways that regulate SASP
The connection between senescent cells and the immune system is mediated by SASP. The inflammation signature of SASP include chemokines, cytokines and other immune modulators. SASP is regulated at multiple levels’ such as, chromatin modification, transcription, translation, mRNA stability and secretion. The autocrine and paracrine positive feedback loops also regulate the signaling of SASP. Most senescent cells express multiple cytokines such as IL-8, CCL2 (MCP-1), CCL7 (MCP-3), CCL8 (MCP-2), CCL13 (MCP-4), CCL3, CCL16, CCL20, CXCL1, CXCL2, CXCL3 ect[35,36]. The most characterized SASP components include multiple pro-inflammatory cytokines, such as interleukin-1 (IL-1), and interleukin-6 (IL-6). IL-1 and IL-6 produced in senescent epithelial and fibroblast cells are capable of inducing cellular senescence in adjacent cells. SASP helps spreading senescence phonotype among nonsenescent cells by creating an inflammatory environment in tissues. The knock-down of IL-6 and IL-8 receptors IL-6R and CXCR2, respectively, prevents senescence.
SASP is induced and regulated by several signaling pathways leading to the activation of the nuclear factor-κB (NF-κB) and/or CCAAT/enhancer-binding protein-β (C/EBPβ). In senescent cells, activated NF-κB and C/EBPβ regulate the expression of SASP factors by controlling, mainly at the transcription level, the inflammatory SASP molecules, IL-1α, IL-6 and IL-8. These inflammatory SASP factors can enhance SASP signaling through activating NF-κB , and C/EBPβ in an autocrine feed-forward fashion[37,38] (Figure 2).
Figure 2 Senescence-associated secretory phenotype signaling pathways.
Nuclear factor kappa light chain enhancer of activated B cells can be activated via multiple signaling pathways such as the GATA binding protein 4, cyclic GMP-AMP synthase-stimulator of interferon genes, and nicotinamide adenine dinucleotide -nicotinamide phosphoribosyltransferase NAD+-NAMT pathways, which lead to the expression of senescence-associated secretory phenotype (SASP) proteins. SASP can be positively regulated through C-X-C motif chemokine receptor 2, or negatively by NOTCH via CCAAT-enhancer-binding proteins. SASP can induce senescence in both autocrine and paracrine manners. SASP can be anti-tumorigenic in the early phase of senescence, but can be pro-tumorigenic in the late phase of senescence. NF-κB: Nuclear factor kappa light chain enhancer of activated B cells; GATA4: GATA binding protein 4; cGAS-STING: Cyclic GMP-AMP synthase-stimulator of interferon genes; SASP: Senescence-associated secretory phenotype; CXCR2: C-X-C motif chemokine receptor 2; C/EBP: CCAAT-enhancer-binding protein.
NOTCH signaling pathway: This pathway has been implicated as an important regulator of SASP. Works from Hoare et al suggested a global upregulation of NOTCH1 accompanied by dynamic alterations of its downstream activity in senescence. They also proposed NOTCH1 as a master regulator of SASP composition via a temporal and functional switch between two different secretomes, TGFβ and pro-inflammatory cytokines through down-regulation of C/EBPβ.
The NOTCH1 was shown to be upregulated in cells undergoing oncogene-induced senescence (OIS), which caused upregulation of TGFβ in the first phase of senescence. The inhibition of NOTCH signaling substantially reduced TGFβ induction in OIS cells. The inhibition of TGFβ in NOTCH1-driven senescent cells prevented the upregulation of the TGFβ-targeted cell cycle inhibitor, p15INK4B. In addition, Hoare et al found that NOTCH1 negatively regulated C/EBPβ but had no effect on NF-κB pathway in the later phase of senescence. The overexpression of activated Notch1 receptors (N1ICD - NOTCH1 intracellular domain) inhibited the ability of C/EBPβ to induce IL-1, IL-6 and IL-8. Moreover, Hoare et al found that inhibiting Notch signaling accelerated clearance of senescent cells in the liver. Therefore, NOTCH signaling pathway may be associated with a complicated mechanism of SASP regulation. But the key questions that remain to be addressed are: (1) Which factor(s) involved in the switching from NOTCH1-TGFβ to NOTCH1-C/EBPβ; (2) What the cross-talk is between NOTCH1-mediated C/EBPβ and NF-kB regulation in senescent cells.
cGAS-STING pathway: cGAS (cGMP-AMP synthase) is a cytosolic DNA sensor and it is activated in response to cytosolic DNA. This activated cGAS binds and activates Stimulator of Interferon Gene (STING). During cellular senescence, the organization of chromatin changes and undergoes degeneration. The integrity of the nuclear envelope is disrupted due to the loss of the nuclear lamina protein Lamin B1. Eventually, small chromatin fragments migrate from nucleus to cytoplasm to become cytoplasmic chromatin fragments (CCF) in senescent cells. cGAS can be activated by any double-stranded DNA irrespective of the sequence. Therefore, CCF from the senescent cells can activate cGAS-STING pathway and upregulate SASP expression via inducing type-1 interferons (IFN-1). The cGAS-STING signaling pathway drives the production of inflammatory SASP cytokines through type I interferon via IRF3 and proinflammatory responses via NF-κB[40,42], thereby facilitating senescence. Gluck et al demonstrated that cGAS deficiency in the liver abrogated the induction of senescence in hepatocytes, which suggests the importance of cGAS in the process of senescence in vivo.
Cecco et al. showed that in oncogene-induced senescence and stress-induced premature senescence, activation of retrotransposable element, L1, and IFN-1 occur in the late phase. They found three factors that caused L1 activation, RB1, FOXA1 and TREX1. RB1 expression was declined in senescent cells but was enhanced in proliferating cells. Upregulation of FOXA1 and downregulation of RB1 and TREX1 were found to be associated with L1 activation and stabilization. The activation of L1 ensured a strong activation of INF-1 response in senescent cells. In addition, when the cGAS-STING pathway was disrupted, the INF-1 response was inhibited and SASP response such as the induction of CCL2, IL-6, and MMP3 were downregulated in the late phase of senescent cells.
NAD+-NAMT pathway: NAD+ and the rate-limiting enzyme of its synthesis, nicotinamide phosphoribosyltransferase (NAMPT), have a critical role in ageing and cancer[45-47]. Recently, Nacarelli et al demonstrated a mechanism of SASP regulation that involves these factors and HMGA proteins HMGA1 and HMGA2. HMGA proteins are known to regulate the chromatin structure and to promote senescence. Now, these proteins are found to regulate NAMPT gene expression by binding at the enhancer element during oncogene-induced senescence of fibroblasts. NAMPT upregulates the expression of proinflammatory SASP fators such as IL-1β, IL-6 and IL-8, which are dependent on the enzymatic activity of NAMPT. This proinflammatory activity are regulated by increased NAD+/NADH ratio. Nacarelli et al have also demonstrated that increased NAD+/NADH ratio causes suppression of AMPK, which interferes with p53-mediated inhibition of p38 MAPK. p38 MAPK controls the transcriptional activity of NF-κB by regulating acetylation of p65. The NF-κB mediated proinflammatory activity is therefore enhanced.
In addition to HMGA proteins, another high mobility group protein, HMGB1, has been implicated in the senescence and SASP. Davalos et al reported the existence of a relationship between HMGB1 and senescence. They found that altered expression of HMGB1 induced senescence in a p53-dependent manner in fibroblasts. Exogenous HMGB1 treatment activated NF-κB activity and IL-6 secretion whereas depletion of HMGB1 attenuated the senescence phenotype. The disruption of normal HMGB1 levels can induce a p53-dependent cell cycle arrest. Interestingly, secreted HMGB1 is essential for optimal secretion of IL-6 and MMP-3, both are important SASP components.
Other pathways: The other known regulators of SASP are bromodomain containg protein 4 (BRD4), lysine methyltransferase MLL1 and G9A. The recruitment of BRD4 to senescence-activated enhancers located adjacent to SASP genes is probably required to induce SASP. But the upstream signaling that regulate these transcriptional activators are not completely known. SASP helps spreading senescence phonotype among nonsenescent cells by creating an inflammatory environment in tissues.
For SASP regulation, mTOR plays an important role as well. The 4E-BP1 is phosphorylated by mTOR. It is a repressor of translation of mRNA. In order to regulate SASP, 4E-BP1 represses IL-1α and MAP kinase-activated protein kinase 2. The degradation of mRNA of proinflammatory SASP factors is associated with a RNA binding protein, zinc finger protein 36L1 (ZFP36L1); and MAP kinase-activated protein kinase 2 inhibits ZFP36L1. By regulating the stability of SASP mRNA, mTOR regulates SASP in senescent cells.
Autophagy and senescence
Autophagy is a highly regulated cellular program responsible for recycling intracellular proteins and damaged/nonfunctional organelles. Autophagy was found to be activated in senescence in human fibroblasts due to the negative feedback of mTOR pathway. A number of genes (e.g., Ulk3 and LC3) associated with autophagy are upregulated in senescent cells and inhibition of autophagy essential genes like Atg5 or Atg7 caused interference of senescence phenotype including SASP. Reciprocally, Kang et al showed that knockdown of autophagy essential genes such as Atg7, Atg12 or lysosomal associated membrane protein 2 (LAMP2) caused senescence in human fibroblasts. This senescence pathway was found to be ROS and p53-dependent. In contrast, Garcia-Prat et al demonstrated that muscle stem cells maintain a reversible quiescence state and do not enter to senescence when the autophagy mechanism is active. This group showed that the muscle stem cells underwent senescence if autophagy was defective and restoration of autophagy prevented senescence in satellite cells. Therefore, autophagy may both induce and prevent senescence, which may be dependent on cell type or the type of experimental model. However, it is still an open question as to whether autophagy is necessary for senescence to occur or it inhibits the senescence process.
Kang et al reported that a transcription factor, GATA4 played an important role in the cellular senescence mechanism. This group suggests that GATA4 is a senescence and SASP regulator. In normal cells, GATA4 binds to an autophagy adaptor, p62, whichis eliminated by the selective autophagy process. This autophagy process is suppressed when senescence is induced. DNA damage activates ATM and ATR kinases, which inhibit p62-mediated GATA4 degradation. The stabilized GATA4 then activates NF-κB inducers TRAF3IP2 and IL-1A. NF-κB, one of the master regulators of senescence, initiates and maintains SASP, and facilitates senescence. The GATA4 activation is found to be independent of p53 and p16INK4A but is dependent on ATM and ATR kinases. This finding suggests that GATA4 may represent a separate branch of DNA damage repair response that induces SASP and hence favors senescence. Kang et al has suggested a clarification on the argument whether autophagy induces or inhibits senescence. Selective autophagy prevents cells from undergoing senescence by limiting GATA4 level when senescence-inducing stimuli, such as irradiation are given but later general autophagy lead to cellular senescence.
At least, one more member of the GATA family, GATA6, has been implicated in the senescence mechanism. Perlman et al reported that GATA6 overexpression could upregulate the expression of p21Cip1 so that the proliferation of vascular smooth muscle cells and fibroblasts were inhibited. Zhang et al suggested the existence of a direct relationship between autophagy and GATA6. Autophagy decreased accumulation of GATA6 and p62 was found to be a negative regulator of GATA6 accumulation. Zhang et al also determined the relationship between an antimalarial drug, dihydroartemisinin (DHA) and GATA6, and their contribution to the hepatic senescence. DHA treatment enhanced the expression of p53, p21Cip1 and p16INK4A and induced senescence in hepatic stellate cells (HSCs) in rat livers. GATA6 accumulation further accelerated the expression of p53 and p16INK4A in DHA-treated livers and promoted senescence. The knock-down of GATA6 by siRNA significantly reduced DHA-induced upregulation of p53 and p16INK4A, and thereby the senescence.