Atypical senescent states: TGFβ-induced senescence and Developmentally programmed senescence

TGFβ-induced senescence: A growing body of evidence suggests that the members of the transforming growth factor beta (TGF- β) family can induce a senescence-like state. Experimentally, senescence has been predominantly, but not exclusively, characterized by the presence of senescence-associated beta galactosidase (SA-β-Gal) staining and the up-regulation of cyclin dependent kinase inhibitors (CDKi) (see below). Human prostate basal cells treated with TGF-β1/2/3 show increased SA-β-Gal activity, which is associated with the flattened, and enlarged cell morphology typical of adherent senescent cells in vitro (Untergasser et al. 2003). Similarly TGF-β1 has been reported to induce a senescent state in bone marrow mesenchymal stem cells as a result of increased mitochondria ROS production (Wu et al. 2014). These cells also showed SA-β-Gal staining and an increased expression of p16. Yu et al. (2010) demonstrated that TGF-β2 could induce a senescent-like state in human trabecular meshwork cells. Again, this was associated with SA-β-Gal staining, increased levels of p16 at both the message and protein level and a reduction in the level of pRB protein. No impact on p21 mRNA or protein expression was observed in response to TGF-β2 exposure. Other groups have also reported a role for TGF-β signaling in inducing a senescent state (Senturk et al. 2010, Minagawa et al. 2011, Acosta et al. 2013). 

It is generally accepted that SA-β-Gal staining should be used in conjunction with several other senescent markers, as it does not appear to detect senescent cells specifically (Severino et al, 2000). However, other than the expression of CDKi, it appears that the phenotypes of cells induced to enter senescence by exposure to TGF-βs have been poorly characterized, especially in regard to immunogenic conversion. Some cell types that become senescent via this route may be cleared by the immune system in a manner analogous to those undergoing developmentally programmed senescence. Others may not and this area represents a fruitful field for further investigation.

Developmentally programmed senescence: Cells sharing features of senescence have been reported within the mesonephros and the endolymphatic sac of the inner ear in human and mouse embryos; as well as the neural roof plate and apical ectodermal ridge in rodents (Munoz-Espin et al. 2013, Storer et al. 2013). The authors hypothesize that this “developmental senescence” (DS) is a programmed part of normal embryonic development. DS was demonstrated experimentally by the presence of SA-β-Gal activity and senescence associated heterochromatin (Munoz-Espin et al. 2013). These cells seem to lack detectable DNA damage and appear to have become senescent independent of p53 and p16 and have gene expression patterns that significantly overlap with those of IMR90 fibroblasts in a state of oncogene-induced senescence. Arrest in this instance is dependent instead upon p21, regulated via the TGF-β/SMAD and PI3K/FOXO pathways (thus showing some affinity with other TGF-β induced senescent states). Interestingly, DS cells are removed during normal embryonic development by macrophages in a manner related to immune clearance of senescent cells in the mature organism (or by apoptosis should senescence fail) contributing to the formation of normal tissue architecture. Thus, the long-recognized distinction between programmed cell death in development and apoptosis in the mature organism appears to be mirrored in DS. Given that the expression of p21 in developing embryos is often attributed to ‘terminal differentiation’ (Vasey et al. 2011), it will be interesting to determine how many of these p21 positive cells are senescent cells and have undergone immunogenic conversion.

Taken from: Cellular Senescence: From Growth Arrest to Immunogenic Conversion

Immune Ligand Expression in Senescent Cells

In addition to secreting soluble factors for the attraction of immune cells, senescent cells can also become immunogenic through the up-regulation of ligands that can specifically be recognized by immune cells.  While research into the recognition and interaction of immune cells with senescent cells is at its infancy, a number of studies have reported the up-regulation of the Natural Killer Group 2D (NKG2D) ligands in senescent cells that can be recognized by receptors on Natural Killer (NK) cells and CD8+ T-cells.  Since NKG2D ligands are not widely expressed on healthy cells, this would allow for specific recognition, interaction and elimination of senescent cells by immune cells.  As with the senescent secretome, this response is likely not exclusive to cell senescence as the same mechanism functions in immunosurveillance of tumour cells (López-Soto et al. 2014).  The human NKG2D ligands primarily consist of MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5 and ULBP6.  The transcriptional up-regulation of MICA and ULBP2 during cell senescence have been reported in senescent activated hepatic stellate cells, replicative senescent fibroblasts and HUVECs, etoposide-induced senescent fibroblasts, fusion-induced senescent fibroblasts and chemotherapy-induced senescent multiple myeloma cells (Krizhanovsky, et al. 2008, Kim et al. 2008 Chuprin et al. 2013, Soriani et al. 2014, Lackner et al, 2014).  In addition to MICA and ULBP2, microarray analysis of replicative senescent fibroblasts demonstrated an increase in the expression of ULBP1 (2.75 fold) compared to growing cells, in addition to the up-regulation of HLA-E (2 fold) (Lackner et al. 2014).  HLA-E is a non-classical MHC class I molecule that plays a role in cell recognition by NK cells. However, replicative senescent vascular smooth muscle cells do not appear to up-regulate MICA, ULBP2 or ULBP1, at least not greater than 2 fold as assessed by microarray analysis (Burton et al. 2009).  Therefore, it should not be assumed that all senescent cell types up regulate NKG2D ligands and this should be evaluated in underexplored senescent cell types. Mechanisms involved in the interaction of senescent cells with T-cells is less understood, but it appears that major histocompatibility complex class II (MHCII) expression is required for killing of pre-malignant senescent hepatocytes by T-cells (Kang et al. 2011).  Mice with liver specific MHCII deficiency resulted in impaired immunosurveillance of senescent cells.

At the mechanistic level, little is currently known about the regulation of NKG2D ligand expression in senescent cells.  Nonetheless, some extrapolation from others models is possible.  For example, MICA and MICB have been reported to be regulated by endogenous miRNAs in tumours and as a result of infection with cytomegalovirus (Stern-Ginossar et al. 2008).  Since miRNAs appear to play a role in regulating cellular senescence (Feliciano et al. 2011, Liu et al. 2012 Benhamad et al. 2012) and their expression is altered in response to DNA damage (Dolezalova et al. 2012, Wang and Taniguchi, 2013), it is possible that changes in miRNA expression also regulate the expression of immune ligands in senescent cells. 

Soriani et al demonstrated that the up-regulation of MICA in senescent multiple myeloma cells was dependent upon the DDR (Soriani et al. 2014).   In other systems, NKG2D ligands have also been shown to be up-regulated in response to DNA damage and Ras activation via ATM and ATR (Gasser et al. 2005, Cerboni et al. 2014).  Inhibition of the ATM or ATR pathways prevented the up-regulation of immune ligands. 

It is also possible that the up-regulation of immune ligands on senescent cells is mediated via the secretory response.  In addition to activating and attracting immune cells, the senescent secretome may serve to up-regulate immune ligands in an autocrine or paracrine manner.  It has been shown for example, that TNFα can up-regulate MICA on human endothelial cells and that the addition of exogenous MICA seems to induce senescence in HUVECs (Lin et al. 2011), but the extent to which this occurs under more physiologically reflective situations remains unclear. 

Immune ligands can also be up-regulated in response to various other forms of cell stress such as heat shock, metabolic stress and endoplasmic reticulum (ER) stress (Cerwenka, 2009, Valés-Gómez et al. 2008).  Thus, as with the secretory response, mechanisms exists that can up-regulate immune ligands independent of DNA damage.  Given that this is an important aspect of senescent cell clearance and the number of cell types in which the up-regulation of immune ligands has been shown is limited, a more detailed study of this aspect of immunogenic conversion seems warranted.

While senescent cells are likely eliminated by the immune system during normal physiological processes, it has been speculated that the accumulation of senescent cells with age could be due to inefficient elimination by an ageing immune system (Burton, 2009).  In fact, immune cells may themselves undergo cellular senescence, a process that requires further investigations (Effros et al. 2005, Rajagopalan et al. 2012). As such, induction of cell senescence in immune cells may represent one aspect of immunosenescence, the gradual deterioration of the immune system, which consequently leads to impaired immunosurveillance of non-immune senescent cells.  It can be speculated that impaired immunosurveillance may result from altered expression of surface receptors on immune cells that impair recognition and interaction with target senescent cells (and cancer cells).  In addition, it is possible that aged or senescent immune cells do not respond as efficiently to chemoattractants secreted by senescent cells.  In order to understand the mechanisms associated with age-related changes resulting in impaired immunosurveillance of senescent cells, we must first fully understand the normal processes governing immune clearance of senescent cells.  However, evaluating the hypothesis that aged or senescent immune cells display a reduced capacity to target senescent cells and the physiological impact of this decline can still be assessed.  If this were indeed found to be the case, the rejuvenation of an ageing immune system would represent an attractive approach for promoting health span.

Apoptosis Resistance in Senescent cells


Senescent cells are frequently referred to as ‘apoptosis resistant’.  This apparent resistance to an apoptotic stimulus in vitro was originally reported by Wang (1995) who observed that late passage (58 population doubling) WI38 fibroblasts were resistant to death caused by serum withdrawal compared to WI38 cultures at less than 15 or approximately 38 population doublings.  All of these human cell populations were dramatically more resistant to death by growth factor deprivation than Swiss 3T3 fibroblasts.  This death resistant phenotype was linked to maintenance of Bcl2 protein levels in senescent WI38 cells.  Subsequent studies extended the resistance phenotype to treatment with both UV light (120mJ) and staurosporin (35nM) and linked it to reduced expression of caspase 3 (Marcotte et al. 2004).  Subsequent work (Ryu et al. 2007) using human dermal fibroblasts confirmed resistance to staurosporin-induced cell death and demonstrated significant resistance to thapsigargin (up to 700nM).  The enhanced survival of senescent dermal fibroblasts under these conditions was attributed to a failure to down regulate Bcl2 under conditions of cellular stress. 

It has been proposed that resistance to apoptotic cell death is a feature of the senescent phenotype that may promote their persistence in vivo, thereby favoring immune clearance over cell death. However, key questions around this phenotypic aspect remain and may be summarized as (i) what are the primary molecular players driving apoptosis resistance in senescent human dermal and lung fibroblasts? (ii) is this phenomenon a general one across tissues and between species?

It is possible that the pro-survival response observed in fibroblasts normally facilitates DNA repair, but is maintained when persistent DNA damage activates the senescent program.  For example, when low levels of DSBs are present, ATM and ATR can result in ERK/NFkB pro-survival signaling (Khalil et al. 2010, Hawkins et al. 2011, Janssens and Tschopp, 2006) that has been associated with the induction of senescent cells by various triggers.  Paradoxically ATM-deficient human fibroblasts are significantly more resistant to cell death triggered by exposure to doxorubicin or low dose ionizing radiation than wild type controls (Park et al. 2012).  However, the population doublings levels of the wild type and mutant cultures were not reported.   If significantly different, this has the potential to confound studies of this type (since normal fibroblast cultures are mixtures of senescent and proliferating cells, the proportions of which alter as the culture is passaged).

In addition to activating cell cycle arrest in response to DNA damage, the p53/p21 pathway can also initiate a pro-survival response.  In some studies, p21 has been shown to play a role in cell survival through its cytoplasmic localization, rather than its nuclear localization associated with cell cycle arrest (Gartel and Tyner, 2002, Piccolo and Crispi 2012, Kreis et al. 2014).  Interestingly, p21 has been reported to be a negative regulator of p53-mediated apoptosis (Gartel and Tyner, 2002), a known response reported in senescent fibroblasts (Seluanov et al. 2001).  p21 has also been reported to promote cell survival in response to oxidative stress by integrating the DDR with endoplasmic reticulum (ER) stress signaling (Vitiello et al. 2009).  However, the up-regulation of p21 may also be required for cells to enter and maintain quiescence (Perucca et al. 2009), suggesting a pro-survival response may occur independent of DNA damage, but dependent upon growth state. 

Autophagy is another feature of senescent cells which can also be initiated by DNA damage and promote cell survival (Rodriguez-Rocha et al. 2011, Singh et al. 2012).  Autophagy promotes cell survival by the degradation of damaged cellular components (Codogno and Meijer, 2005), probably as a result of elevated ROS (Scherz-Shouval and Elazar, 2011) in the case of cell senescence.  Interestingly, there is crosstalk between autophagy and apoptosis pathways (Zhou et al. 2011, Xu et al. 2013, Lindqvist and Vaux, 2014), with particular emphasis on the anti-apoptotic Bcl2 protein family.   

It has long been recognized that cytokines and their binding proteins can act to modulate cell survival (Lotem and Sachs, 1999).  Given the altered secretory phenotype of some senescent cells, it would be unsurprising if this did not contribute to altered death dynamics, but the mechanisms by which this could occur are potentially highly complex.   For example Interleukin-6 (secreted by senescent cells) has been shown to promote cell survival in transformed cells (Biroccio et al. 2013), and its secretion by cancer-associated fibroblasts protects luminal breast cancer cells from tamoxifen treatment (Sun et al. 2014).  Whilst inhibition of insulin-like growth factor-1 (IGF-1) has been shown to induce apoptosis in senescent fibroblasts (Luo et al. 2014), the alteration of IGF-1 binding proteins are just as likely to influence cell survival.  For example, insulin-like growth factor binding protein 3 (IGFBP-3) is both transcriptionally up-regulated and secreted in elevated amounts by senescent human fibroblasts (Hampel et al. 2005).  IGFBP-3 triggers enhance apoptotic cell death in tumor cells when internalized and translocated to the nucleus, where it targets intracellular regulators of apoptosis (Hampel et al. 2005). Endocytotic uptake of IGFBP-3 in senescent human fibroblasts did not occur.  This has the potential to render them apoptosis resistant and capable of promoting apoptosis in cells nearby.  It could be speculated that in a microenvironment characterized by high cell turnover, both senescent and precancerous cells could be in close proximity. Elevated local IGFBP-3 generated by senescent cells could thus act as a paracrine tumour suppression mechanism.  This idea remains untested.

It seems doubtful that global apoptosis resistance is a general feature of senescent cells.  For example, early work by one of us (RGAF) failed to show any elevation in spontaneous apoptosis rates in HUVECs cultured to senescence (although baseline apoptosis rates as measured by TUNEL were significantly higher than those seen in fibroblasts) (Kalashnik et al. 2000).  Later studies (Hoffman et al. 2001) demonstrated that late passage HUVECs were more sensitive to apoptosis induced by oxidized LDL or TNFα compared to early passage cells.  Jeon and Boo (2013) have recently shown that up-regulation of the Fas receptor at both the mRNA and protein level in senescent HUVECs probably underlies their enhanced potential to undergo programmed cell death.  Perhaps most compellingly, Hample et al. (2004) demonstrated in parallel culture experiments that whilst senescent human dermal fibroblasts were more resistant to cell death induced by exposure to ceramide than early passage cells, senescent HUVECs were significantly more apoptosis prone.

It is interesting that minimal changes in baseline apoptosis rates could be detected in senescent HUVEC populations despite their increased sensitivity to Fas or ceramide-induced killing.  However Wang et al. (2004) reported an analogous phenomenon in senescent human keratinocytes.  This study demonstrated that spontaneous apoptosis rates did not alter in cultures of senescent human keratinocytes (duplicating an earlier report by Norsgaard et al. 1996).  Nonetheless, levels of Fas and related apoptotic effectors (e.g. FLICE) increased whilst Bcl2 declined significantly (as measured by ELISA).  The authors showed that antibody-mediated Fas activation or medium exhaustion increased the apoptotic fraction from 3-5% to 30% in senescent keratinocytes, whilst leaving apoptosis levels unchanged in early passage cultures.

Interestingly, Crescenzi et al. (2011) have recently shown that induction of premature senescence in human cancer cell lines also induces Fas expression, and concomitant susceptibility to Fas-induced apoptosis.  Fibroblasts rendered senescent by serial passage are also susceptible to Fas-mediated killing (Tepper et al. 2000).  Thus it is possible that at senescence, human cell types differ in their resistance to apoptosis induced by stressors, but show a common susceptibility to Fas/TNFα mediated killing.  If immunogenic conversion were a key hallmark of senescence, then this would seem plausible.  It does however require significant additional experimental study.

As with the secretory response, it should not be assumed that an “apoptosis resistant” phenotype is conserved across species.  For example Mayogora et al. (2004) demonstrated that cultures of cardiac fibroblasts from Sprague-Dawley rats were more resistant to apoptosis induced by serum withdrawal or staurosporin, than dermal fibroblast cultures initiated from the same animals.  Dermal fibroblasts from this species apparently lacked Bcl2 protein as measured by Western blot (although it remained readily detectable in cardiac fibroblasts).  This is a clear species difference and suggests that researchers working in other systems should not assume that the features observed in human cells are duplicated across the animal kingdom.

Cellular senescence: from growth arrest to immunogenic conversion

Abstract


Cellular senescence was first reported in human fibroblasts as a state of stable in vitro growth arrest following extended culture. Since that initial observation, a variety of other phenotypic characteristics have been shown to co-associate with irreversible cell cycle exit in senescent fibroblasts. These include (1) a pro-inflammatory secretory response, (2) the up-regulation of immune ligands, (3) altered responses to apoptotic stimuli and (4) promiscuous gene expression (stochastic activation of genes possibly as a result of chromatin remodeling). Many features associated with senescent fibroblasts appear to promote conversion to an immunogenic phenotype that facilitates self-elimination by the immune system. Pro-inflammatory cytokines can attract and activate immune cells, the presentation of membrane bound immune ligands allows for specific recognition and promiscuous gene expression may function to generate an array of tissue restricted proteins that could subsequently be processed into peptides for presentation via MHC molecules. However, the phenotypes of senescent cells from different tissues and species are often assumed to be broadly similar to those seen in senescent human fibroblasts, but the data show a more complex picture in which the growth arrest mechanism, tissue of origin and species can all radically modulate this basic pattern. Furthermore, well-established triggers of cell senescence are often associated with a DNA damage response (DDR), but this may not be a universal feature of senescent cells. As such, we discuss the role of DNA damage in regulating an immunogenic response in senescent cells, in addition to discussing less established “atypical” senescent states that may occur independent of DNA damage.




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Cell Senescence: From Physiology to Pathology
Santiago de Compostela, Spain
19-22 July 2015

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Physiological impact of cell senescence in vivo: Limiting Tissue Damage


In addition to their tumor suppression function, senescent cells also play a beneficial role in non-cancer pathology by limiting tissue fibrosis.   For instance, tissue damage within the liver stimulates the activation of hepatic stellate cells (HSCs), which hyper- proliferate and secrete extracellular matrix components to form a fibrotic scar.  Hyper- proliferation of HSCs induces cell senescence leading to a reduction in the secretion of ECM proteins and enhanced secretion of ECM degrading proteins, thereby limiting fibrosis.  Senescent HSCs are then eliminated in a timely manner by immune cells such as natural killer (NK) cells.  When the mechanisms leading to NK cell mediated elimination are disabled, fibrosis is increased.  In mice lacking molecular components required for induction of cell senescence, HSCs continue to proliferate, depositing ECM components and elevating the fibrotic response.  Therefore, induction of senescence in HSCs prevents short-term tissue damage by limiting fibrosis.  In addition to the liver, a similar process occurs during tissue repair within the pancreas by senescent pancreatic stellate cells.   In this instance, it was suggested that lymphocytes at the sites of wounds might play a duel-specific role in pancreatic fibrogenesis by triggering both the initiation of wound healing by activating stellate cells and its completion by clearance of senescent stellate cells.


Cell senescence also limits tissue damage at sites of cutaneous wound healing, where secretion of CCN1 induces fibroblast senescence associated with an elevation in the DNA damage response and the activation of p53 and RAC1-NOX1 complex.  The expression of anti-fibrotic genes by CCN1-induced senescent cells prevented excess fibrosis, whereas mice that express a senescence-defective Ccn1 mutant resulted in elevated fibrosis.    CCN1 also appears to play a role in the regression of liver fibrosis through induction of cell senescence in HSCs.  Therefore, cell senescence is a mechanism that limits tissue damage in multiple tissues and serves not only to restrain the damage, but also to initiate the repair and return the tissue to the pre-damaged state.

The main focus of ageing research is to prevent/combat age-related disease and disability, allowing everyone to live healthier lives for longer.