Atypical senescent states: Endoplasmic Reticulum stress induced senescence

Endoplasmic reticulum (ER) stress may also promote a senescent-like response.  The accumulation of unfolded proteins in the ER triggers a stress-signaling pathway that can result in cell cycle arrest mediated by p27 (Han et al. 2013) and the p53/47 isoform (Bourougaa et al. 2010).  Furthermore, ER stress has also been shown to induce an inflammatory response via NFkB activation (Garg et al. 2012) and induce cytokines such as MCP-1, IL-6 and IL-8 (Schroder, 2008), which are capable of attracting and activating immune cells (Sagiv and Krizhanovsky, 2013). ER stress has also been shown to promote cell survival, another feature of cell senescence (Raciti et al. 2012).  Interestingly, a senescent state via activation of ER stress-dependent p21 signaling has been reported in proximal tubular epithelial cells, triggered by receptors for advanced glycation end-products (RAGE) (Liu et al. 2014).  Although, ER stress-induced senescence has the potential induce an immunogenic phenotype in the absence of DNA damage, a full evaluation of the phenotype is required to determine if this is so.

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

Quiescent to Senescent Conversion

The presence of DNA damage and subsequent up-regulation of p16(INK4a) in quiescent cells in vivo may induce a pre-senescent state that converts to a full senescent state when cells are stimulated to proliferate. This suggests that DNA replication is required to induce a persistent DDR associated with cell senescence.   For example, damage to skeletal muscle in normal young mice causes the activation of quiescent satellite cells (adult stem cells), which proliferate and undergo myogenic differentiation required for muscle repair. However, a recent study has shown that in geriatric mice (28-32 months of age), satellite cell activation is impaired and satellite cells instead convert from a pre-senescent state (quiescent cells with high p16(INK4a) expression) to a full senescent state (including a DDR) when stimulated to proliferate in response to injury. As such, the induction of senescent satellite cells with age can impair satellite muscle regeneration. This study suggests that senescent cells may accumulate in late life due to a conversion from quiescence to senescence (termed geroconversion) in response to a requirement for cells to replicate over time to regenerate tissue. In this model, more and more quiescent cells are likely to accumulate DNA damage over the life-time of an organism and are therefore more likely to become senescent when induced to proliferate later in life. Therefore, if quiescent cells inflicted with DNA damage convert to senescence when stimulated to proliferate, then eliminating such damage may prevent this conversion.

Geriatric muscle stem cells switch reversible quiescence into senescence

Physiological and pathological consequences of cellular senescence

Reclassifying Cellular Senescence into Two Main Types

Persistent activation of the DNA-damage response (DDR) can trigger cells to undergo cellular senescence, a state of irreversible, immune evoking, growth arrest.  In such a way, cellular senescence can prevent tumourigenesis firstly by blocking cells from replicating and producing abnormal and potentially cancerous daughter cells and secondly by coordinating their removal by immune cells.  Additionally, senescent cells can aid tissue repair by preventing extensive cellular proliferation leading to fibrosis, possibly triggered by replication stressed-induced DNA damage.  However, if this orchestrated removal of senescent cells becomes dysregulated, then persistent senescent cells can promote tumourigenesis and tissue damage.

An aspect of the DDR in senescent cells is the induction of an array of secretory factors, including cytokines/chemokine’s, which are important in attracting/activating immune cells to their vicinity.  When immune cells reach the locality of senescent cells, they can then specifically recognize them by the expression of immune ligands on the cell membrane, a process that may also be regulated primarily by the DDR.  The specific ligands recognized and the mechanism of senescent cell death will then be dependent upon the type of immune cell interacting with the senescent cell. 

However, cells induced to undergo permanent growth arrest in vitro by the overexpression of the cyclin dependent kinase inhibitor p16ink4a, do not develop an immune evoking secretory phenotype until the addition of DNA damage.   If cells in physiological or pathological conditions can indeed undergo permanent cell cycle arrest in a p16 dependent, DDR-independent manner, then these cells are unlikely to evoke an immune response for their clearance.  In support of this, a recent study demonstrated that cells overexpressing p14(ARF) in the epidermis of mice remained present for weeks after transgene silencing (Tokarsky-Amiel et al 2013).  Even if p16-induced senescent cells do not display a pro-inflammatory phenotype, they can still cause physiological problems simply by their inability to proliferate, an essential feature required for tissue regeneration and maintenance.  In this regard, the growth arrest and pro-inflammatory phenotype of senescent cells can be investigated separately to determine which feature is important in different physiological contexts. 

Until the phenotype of p16-induced senescent cells in vivo have been researched more extensively, cellular senescence could be divided into two separate types.  Firstly, immunogenic senescence related to a DNA damage response, consisting of a pro-inflammatory phenotype and the presence of immune ligands, triggered by telomere shortening, oncogene-activation, and chemical stressors.  Secondly, sterile senescence which lacks a pro-inflammatory phenotype and the inability to evoke an immune response. 

If this distinction is made, then studies focused on the effect of cellular senescence on ageing, disease and cancer development can better design their experiments and avoid confusion between conflicting results due to differences in the types of senescence used.  

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Clearance of p16Ink4a-positive senescent cells delays ageing-associated disorders

A paper worth reading. Excellent work.


ABSTRACT

Darren J. Baker, Tobias Wijshake, Tamar Tchkonia, Nathan K. LeBrasseur, Bennett G. Childs, Bart van de Sluis, James L. Kirkland & Jan M. van Deursen

Advanced age is the main risk factor for most chronic diseases and functional deficits in humans, but the fundamental mechanisms that drive ageing remain largely unknown, impeding the development of interventions that might delay or prevent age-related disorders and maximize healthy lifespan. Cellular senescence, which halts the proliferation of damaged or dysfunctional cells, is an important mechanism to constrain the malignant progression of tumour cells1, 2. Senescent cells accumulate in various tissues and organs with ageing3 and have been hypothesized to disrupt tissue structure and function because of the components they secrete4, 5. However, whether senescent cells are causally implicated in age-related dysfunction and whether their removal is beneficial has remained unknown. To address these fundamental questions, we made use of a biomarker for senescence, p16Ink4a, to design a novel transgene, INK-ATTAC, for inducible elimination of p16Ink4a-positive senescent cells upon administration of a drug. Here we show that in the BubR1 progeroid mouse background, INK-ATTAC removes p16Ink4a-positive senescent cells upon drug treatment. In tissues—such as adipose tissue, skeletal muscle and eye—in which p16Ink4a contributes to the acquisition of age-related pathologies, life-long removal of p16Ink4a-expressing cells delayed onset of these phenotypes. Furthermore, late-life clearance attenuated progression of already established age-related disorders. These data indicate that cellular senescence is causally implicated in generating age-related phenotypes and that removal of senescent cells can prevent or delay tissue dysfunction and extend healthspan.


Link: http://www.nature.com/nature/journal/vaop/ncurrent/full/nature10600.html

Replicative senescence at the molecular level in vitro

Telomere-independent senescence (TIS)

Like p21, p16 is a CDK inhibitor involved in cell cycle arrest. Over expression of p16 causes G1 arrest in early passage cells by inhibiting the phosphorylation of the retinoblastoma protein (Kato et al, 1998). However, the removal of p16 activity results in only minimal lifespan extension that was terminated by senescence (Wei et al, 2003). p16 plays a role in maintaining senescence triggered by a short telomere. It appears that p21 is the initiating factor in the growth arrest observed in senescent cells, but p16 is required to maintain that state. p21 has been shown to initially increase in fibroblast cultures but later gradually decrease (Alcorta et al, 1996). During the decline of p21, p16 protein levels gradually increased in senescent cultures, reaching nearly 40-fold higher than in early cultures.

P16 is thought to trigger senescence independently of telomere length. For example, the inactivated p53 in two different fibroblast strains: WI-38 (from foetal lung) and BJ (from neonatal foreskin) resulted in the continuation of growth without Rb inactivation in BJ cells, whereas WI-38 cells fail to grow even when both Rb and p53 are inactivated. It was suggested that these two outcomes was due to the intrinsic differences in the ability to induce p16 at senescence. This proposal was based on the understanding that like WI-38, other human cells appear to undergo replicative senescence prior to telomere shortening by the induction of p16 (Itahana et al, 2003). The ability of p16 to cause growth arrest even after Rb inactivation also suggests that p16 can function independently of Rb activity. Further support for a telomere-independent mechanism was provided from studies which have shown that telomerase activity alone was insufficient to extend the replicative potential of human keratinocytes or mammary epithelial cells (Kiyono et al, 1998). It was shown that down-regulation of p16 in combination with telomerase activity did lead to replicative lifespan extension. Another study found that cyclin D1 over-expression in primary oral keratinocytes extend the lifespan, whereas the combination of cyclin D1 over-expression and p53 inactivation led to their immortalisation (Opitz et al, 2001). Cyclin D1 forms complexes with CDK4 which subsequently phosphorylates and inactivates retinoblastoma (Rb) growth repression (Connell-Crowley et al, 1997). P16 inhibits cell cycle progression by inhibiting CDK4. It appears that the over-expression of cyclin D1 bypasses p16-TIS resulting in an extension of lifespan. In this instance the p16 mechanism has become redundant and the cells continue to divide until TDS mechanism is activated. This may be why these cells are immortalised by p53 inactivation.

This telomere-independent mechanism may be the primary trigger of cellular senescence in mice and rats. Mice and rats have telomeres which are 5-10 times longer than those of human cells (Shay and Wright, 2001). Despite this increase in length, rodent cells engineered to lack telomerase show telomere shortening with no effect on replicative potential. These cells senesce in the presence of long telomeres. Since mouse and rat cells repair DNA damage far less efficiently than do human cells, it has been suggested that such damage may be the trigger for p16 induction.