Posts Tagged ‘granular exostosis’

Insight on Cell Senescence

Larry H. Bernstein, MD, FCAP, Curator



Granule exocytosis mediates immune surveillance of senescent cells

A Sagiv1 , A Biran1 , M Yon2,3, J Simon2,4, SW Lowe2,4 and V Krizhanovsky1,2
Oncogene (2013) 32, 1971–1977    http://www.nature.com/onc/journal/v32/n15/pdf/onc2012206a.pdf

Senescence is a stable cell cycle arrest program that contributes to tumor suppression, organismal aging and certain wound healing responses. During liver fibrosis, for example, hepatic stellate cells initially proliferate and secrete extracellular matrix components that produce fibrosis; however, these cells eventually senesce and are cleared by immune cells, including natural killer (NK) cells. Here, we examine how NK cells target senescent cells and assess the impact of this process on liver fibrosis. We show that granule exocytosis, but not death-receptor-mediated apoptosis, is required for NK-cell-mediated killing of senescent cells. This pathway bias is due to upregulation of the decoy death receptor, Dcr2, an established senescence marker that attenuates NK-mediated cell death. Accordingly, mice with defects in granule exocytosis accumulate senescent stellate cells and display more liver fibrosis in response to a fibrogenic agent. Our results thus provide new insights into the immune surveillance of senescent cells and reveal how granule exocytosis has a protective role against liver fibrosis. Oncogene (2013) 32, 1971–1977; http://dx.doi.org:/10.1038/onc.2012.206


Senescence is accompanied by phenotypic and transcriptional changes that identify senescent cells in vitro and in vivo. For example, senescent cells display a large and flat morphology in vitro and upregulate a senescence-associated b-galactosidase (SA-b-gal).9 Senescent cells often display global changes in chromatin structure10 that are associated with downregulation of cell cycle genes and components of the extracellular matrix and upregulation of immune modulators and matrix degrading enzymes.4 Comparative analyses of gene expression data have produced some markers that appear specific for senescence,11 including the p15ink4b cyclin-dependent kinase inhibitor and the decoy receptor 2 (Dcr2, formally TNFRSF10D). Although p15ink4b likely contributes to the senescence-associated cell cycle arrest,12 whether decoy receptors or some other senescence markers actively participate in the program remains unknown.

Senescence acts through a coordinated program involving cell autonomous and cell nonautonomous components.13 In a cell autonomous manner, the Rb and p53 tumor suppressor pathways act to produce the stable cell cycle arrest that is the hallmark of senescence.1 These proteins are activated by, or activate, cyclindependent kinase inhibitors, such as p15ink4b, p16ink4a and p21, which lead to stable suppression of E2F target genes.10,14 Secreted proteins, regulated at least partially by NF-kB, enhance cell cycle arrest and are largely responsible for mediating the impact of senescent cells on tissue biology.15–17 These factors can attract immune cells, including natural killer (NK) cells, triggering the recognition and ultimate clearance of the senescent cells from tumors or tissue.4,18 Such mechanisms may be necessary to prevent the long-term damage that might be produced by senescent cells, and to facilitate tissue repair and homeostasis.

The mechanisms whereby NK cells eliminate senescent cells from tissues are not known. NK cells rely on two independent mechanisms to eliminate a variety of external and internal threats, including tumor cells.19,20 The ligands on the surface of NK cells, TRAIL and FAS ligand (FasL) bind corresponding receptors on target cells leading to caspase activation and cell death—a process that can be exquisitely controlled though the expression of various positive and negative regulators.21,22 NK cells can also eliminate target cells through granule exocytosis, a process involving the production of perforin and granzyme (A, B) containing granules, which are secreted from the NK cell upon interaction with the target cell.21,23 Perforin is responsible for perforating the cell membrane and thus enabling granzyme release into the target cells where it can induce cell death by both caspase-dependent and independent pathways.24 B

Here, we set out to understand how NK cells eliminate senescent cells from tissues and the implications of such mechanisms on liver fibrosis. Our results indicate that the granule exocytosis, and not death-receptor-mediated apoptosis, is essential for the NK-mediated surveillance of the senescent cells and that disruption of this pathway leads to the accumulation of senescent cells in damaged livers and increased fibrosis. Our study thus provides the key biological and mechanistic insights into the immune surveillance of senescent cells.

Figure 1. NK cells preferentially recognize senescent cells in a wide range of target:effector cell ratios. Senescent or growing IMR-90 fibroblasts were co-incubated with YT cells for 12 h at the indicated ratios and cytotoxicity was determined. The graphs represent the average and the s.e. of triplicate measurements from at least three independent experiments. *Po0.005.

Figure 2. Caspases are dispensable for NK-mediated cell killing of senescent cells. Senescent or growing IMR-90 fibroblasts were incubated for 12 h with either 2 or 10 nM FasL (a). Caspase inhibitors Z-VAD-FMK or Z-IEDT-FMK were added at the concentration of 10 mM as indicated. Cytotoxicity was determined at the end of the coincubation period. Senescent or growing IMR-90 fibroblasts were coincubated with YT cells for 12 h in the presence of 10 mM of caspase inhibitors Z-VAD-FMK or Z-IEDT-FMK and then the cytotoxicity was determined (b). The graphs represent the average and the s.e. of triplicate measurements from at least three independent experiments. *Po0.05, ***Po0.001.

Figure 3. Granule exocytosis pathway is required for NK-cell-mediated killing of senescent cells. Senescent and growing IMR-90 fibroblasts (a, c) or HSCs (b) were co-incubated with YT cells for 12 h (a, b) or with primary NK cells for 2 h (c). Cytotoxicity assays were performed either in the presence of 100 nM granule exocytosis inhibitor, CMA or following pre-incubation of the YT or primary NK cells with 25 mM Granzyme B inhibitor 3,4-DCI. The graphs represent the average and the the s.e. of triplicate measurements from at least three independent experiments. *Po0.01, **Po0.001, ***Po0.0001.

Figure 4. Dcr2 attenuates killing of senescent cells through the death receptor pathway. Dcr2 expression level in senescent and growing IMR- 90 fibroblasts (a, b) and human HSCs (c, d) were evaluated by quantitative RT–PCR analysis (a, c) and immunoblotting (b, d). Dcr2-deficient senescent IMR-90 cells were incubated with either 10 or 100 ng/ml TRAIL and cytotoxicity was determined (e), and Dcr2 knockdown confirmed (f). Senescent IMR-90 cells with siDcr2 or siControl were incubated with YT cells for 12 h and cytotoxicity was determined (g). In the parallel approach IMR-90 cells were infected with short hairpin RNA (shRNA) targeting Dcr2 (shDcr2) or control shRNA targeting luciferase (shLuci) and induced to senescence by etoposide treatment. Dcr2 protein level was assessed by immunoblot (h). The cells were co-incubated for 12 h with YT cells and cytotoxicity was determined (i). The graphs represent the average and the s.e. of triplicate measurements from at least four independent experiments *Po0.05, **Po0.001, ***Po0.0001.

Figure 5. Perforin promotes senescent cell clearance and limits liver fibrosis. Perforin knockout (Prf / ) and wt mice were treated with CCl4 to induce fibrosis. H&E and Sirius red staining show liver morphology and accumulation of fibrotic scar following the treatment (a). Morphometric analysis of Sirius red stained, entire liver sections (b). Expression of markers of activated HSCs, aSMA and Colagen1a, and senescence marker p15ink4b were tested by immunoblotting of whole-liver extracts (c). Four mice of each genotype are shown. SA-b-gal staining identified accumulation of senescent cells along the fibrotic scar areas in the livers (d). The presence of SA-b-gal-positive cells was quantified in the entire liver sections (e). At least five mice of each genotype were used for the analysis in B and E; **Po0.001, ***Po0.0001.


NK-cell-mediated clearance of senescent cells is one component of the coordinated process whereby cellular senescence limits the extent of liver fibrosis and facilitates wound repair.4,18 Recent studies also suggest that senescent cell clearance by immune cells promotes tumor regression in established tumors.18 Our results demonstrate that the granule exocytosis pathway, but not the death receptor pathway, is necessary for the specific killing of senescent fibroblasts and stellate cells by NK cells and participates in the clearance of senescent activated HSCs to limit liver fibrosis. Therefore, NK-cell-mediated cytotoxicity through granule exocytosis contributes to immune surveillance of senescent cells in vitro and in vivo.

In addition to the granule exocytosis pathway, most cytotoxic lymphocytes engage the death receptor pathway to eliminate target cells. This pathway is widely used by NK cells in the liver.21 NK cell express high levels of the death receptor ligand TRAIL upon activation with IL-2,26 are suggested to participate in the surveillance of the HSCs,35 and protect against tumor development following chemical carcinogenesis.36 Given this, we were surprised that death-receptor-mediated cytotoxicity was dispensable for the immune surveillance of senescent cells. Consistent with these findings, an anti-TRAIL antibody failed to inhibit immune system-mediated tumor clearance following p53 restoration in a liver carcinoma model18 (W Xue and SWL, unpublished data). Of course, we cannot rule out the possibility that death receptor pathways contribute to senescent cell clearance in other settings.

Why does granule exocytosis, and not the death-receptor signaling, mediate NK-cell surveillance of senescent cells? Mechanistically, this appears partly because of the accumulation of Dcr2 during senescence, which occurs in fibroblasts, certain epithelial cells11,18 and, as shown here, also senescent activated HSCs. Dcr2 can bind death-receptor ligands, with higher affinity to TRAIL, but as it lacks the activation domain it prevents downstream signaling through the death receptor pathway31,37 and, therefore, can protect senescent cells from death-receptorligand-mediated killing. Another decoy receptor, Dcr3, has higher affinity to FASL.38 However, in contrast to Dcr2, Dcr3 is a secreted receptor and is much less likely to have a role in direct interaction between senescent and NK cells. Although previously considered merely a senescence marker, our results establish a functional role for Dcr2 in protecting senescent cells from cytotoxicity through the death receptor pathway induced by NK cells and possibly other cells as well. The biological rationale for this regulation remains unclear, but may serve to prevent autoimmunity following short-term tissue damage.

In addition to blocking the death receptor pathway, senescent cells may also stimulate NK cells to induce the perforin-mediated killing. Senescent cells upregulate expression of several ligands of NK-cell receptor NKG2D4,39 and ICAM-1, the ligand of NK-cell receptor LFA-1.40 Studies suggest that activation of the NKG2D receptor induces granule exocytosis to eliminate cancer cells, a process that might be reinforced by signaling from LFA-1.41 In this manner, ligands upregulated in senescent cells might activate multiple NK-cell receptors to trigger granule exocytosis.

The role of granule exocytosis in the surveillance of senescent cells has important ramifications for understanding and treating wound healing and cancer. Indeed, we show that the immune clearance of senescent activated HSCs has a significant impact on the pathophysiology of liver fibrosis in which the granule exocytosis pathway has been previously implicated.42,43 Beyond the liver, immune surveillance of senescent cells might have a significant role in other fibrosis-related pathological conditions.

Still, the most prevalent conditions where senescence has been studied to date involve cancer and aging.3,9 Senescent cells accumulate with age and contribute to functional decline of multiple tissues7,9 while perforin-mediated granule exocytosis diminishes at that time.47,48 Separate studies suggest that the integrity of the granule exocytosis pathway can modulate a variety of cancer phenotypes.49,50 Though definitive proof will require further testing, we speculate that the granule exocytosis pathway contributes to immune surveillance of senescent cells in each of these conditions. In principle, pharmacological modulation of this pathway, as has been recently described using IL21,51 might increase the clearance of senescent cells from premalignant, damaged or aged tissues to limit carcinogenesis and the decline in tissue function accompanying the accumulation of senescent cells.

REFERENCES 1 Serrano M, Lin AW, McCurrach ME, Beach D, Lowe SW. Oncogenic ras provokes premature cell senescence associated with accumulation of p53 and p16INK4a. Cell 1997; 88: 593–602.

2 Schmitt CA, Fridman JS, Yang M, Lee S, Baranov E, Hoffman RM et al. A senescence program controlled by p53 and p16INK4a contributes to the outcome of cancer therapy. Cell 2002; 109: 335–346.

3 Narita M, Lowe SW. Senescence comes of age. Nat Med 2005; 11: 920–922.

4 Krizhanovsky V, Yon M, Dickins RA, Hearn S, Simon J, Miething C et al. Senescence of activated stellate cells limits liver fibrosis. Cell 2008; 134: 657–667.

5 Jun JI, Lau LF. The matricellular protein CCN1 induces fibroblast senescence and restricts fibrosis in cutaneous wound healing. Nat Cell Biol 2010; 12: 676–685.

6 Pitiyage GN, Slijepcevic P, Gabrani A, Chianea YG, Lim KP, Prime SS et al. Senescent mesenchymal cells accumulate in human fibrosis by a telomereindependent mechanism and ameliorate fibrosis through matrix metalloproteinases. J Pathol 2011; 223: 604–617.

7 Baker DJ, Wijshake T, Tchkonia T, LeBrasseur NK, Childs BG, van de Sluis B et al. Clearance of p16Ink4a-positive senescent cells delays ageing-associated disorders. Nature 2011; 479: 232–236.

8 Kang TW, Yevsa T, Woller N, Hoenicke L, Wuestefeld T, Dauch D et al. Senescence surveillance of pre-malignant hepatocytes limits liver cancer development. Nature 2011; 479: 547–551.

9 Dimri GP, Lee X, Basile G, Acosta M, Scott G, Roskelley C et al. A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc Natl Acad Sci USA. 1995; 92: 9363–9367.

10 Narita M, Nunez S, Heard E, Narita M, Lin AW, Hearn SA et al. Rb-mediated heterochromatin formation and silencing of E2F target genes during cellular senescence. Cell 2003; 113: 703–716

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