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              Advocacy & Research for Unlimited Lifespans


Downregulation of the inflammatory network in senescent fibroblasts and aging tissues of the long‐lived and cancer‐resis

cellular senescence dna damage dna repair interleukin‐1 alpha (il1α) nuclear factor κb (nf‐κb) senescence‐associated secretory phenotype (sasp) spalax

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#1 Engadin

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Posted 19 October 2019 - 01:45 PM






F U L L   T E X T   S O U R C E :   Aging Cell - Wiley Online Library







The blind mole rat (Spalax) is a wild, long‐lived rodent that has evolved mechanisms to tolerate hypoxia and resist cancer. Previously, we demonstrated high DNA repair capacity and low DNA damage in Spalax fibroblasts following genotoxic stress compared with rats. Since the acquisition of senescence‐associated secretory phenotype (SASP) is a consequence of persistent DNA damage, we investigated whether cellular senescence in Spalax is accompanied by an inflammatory response. Spalax fibroblasts undergo replicative senescence (RS) and etoposide‐induced senescence (EIS), evidenced by an increased activity of senescence‐associated beta‐galactosidase (SA‐β‐Gal), growth arrest, and overexpression of p21, p16, and p53 mRNAs. Yet, unlike mouse and human fibroblasts, RS and EIS Spalax cells showed undetectable or decreased expression of the well‐known SASP factors: interleukin‐6 (IL6), IL8, IL1α, growth‐related oncogene alpha (GROα), SerpinB2, and intercellular adhesion molecule (ICAM‐1). Apparently, due to the efficient DNA repair in Spalax, senescent cells did not accumulate the DNA damage necessary for SASP activation. Conversely, Spalax can maintain DNA integrity during replicative or moderate genotoxic stress and limit pro‐inflammatory secretion. However, exposure to the conditioned medium of breast cancer cells MDA‐MB‐231 resulted in an increase in DNA damage, activation of the nuclear factor κB (NF‐κB) through nuclear translocation, and expression of inflammatory mediators in RS Spalax cells. Evaluation of SASP in aging Spalax brain and intestine confirmed downregulation of inflammatory‐related genes. These findings suggest a natural mechanism for alleviating the inflammatory response during cellular senescence and aging in Spalax, which can prevent age‐related chronic inflammation supporting healthy aging and longevity.
Ten years have passed since senescence‐associated secretory phenotype (SASP), a complex of predominantly pro‐inflammatory factors secreted by aging cells, was first described (Coppe et al., 2008; Kuilman & Peeper, 2009). The seminal works on this topic heralded a new era of understanding the involvement of cellular senescence in age‐related pathology. Senescent cells lose their ability to divide, inhibiting the propagation of mutations to the next cell generation in response to telomere shortening, DNA damage, or oncogenic stimulus. Notwithstanding the physiological role of senescence in preventing malignant transformation (Campisi, 2013), senescent cells accumulate in damaged or aging tissues and influence the surrounding cells through SASP factors (Lasry & Ben‐Neriah, 2015). SASP contributes to tissue repair and reinforcement of senescence in damaged cells (Acosta et al., 2013); however, it is involved in the maintenance of the pro‐inflammatory microenvironment, resulting in the acquisition of sterile inflammation, leading to age‐associated diseases including cancer (Franceschi & Campisi, 2014; Tchkonia, Zhu, van Deursen, Campisi, & Kirkland, 2013).
The DNA damage response (DDR), the main inducer of SASP, is activated in response to DNA lesions that trigger DNA repair programs (Rodier et al., 2009). If DNA damage is not resolved, cells undergo a nonreversible proliferative arrest associated with a persistently active DDR, causing the secretion of inflammatory factors. Data indicate that detrimental effect of senescent cells on surrounding tissues and the whole body is attributed to both the effects of arrested cells themselves (McGlynn et al., 2009) and secretion of SASP factors (Tchkonia et al., 2013). Therefore, inhibition of SASP is now considered an alternative to senolytic therapy to target the deleterious effects of senescent cells (Georgilis et al., 2018; Tchkonia et al., 2013).
Recent evidence has indicated that SASP and cell cycle arrest are regulated by different signaling molecules. It was demonstrated that senescence may occur without genotoxic stress, for example, by overexpression of p16Ink4a, the regulator of retinoblastoma protein (Coppe, Desprez, Krtolica, & Campisi, 2010). The secretion of interleukin‐6 (IL6), a key SASP factor, was shown to be unrelated to senescence arrest, but rather caused by persistent DDR activation in a p53‐independent manner (Rodier et al., 2009). Rapamycin, the inhibitor of mammalian target of rapamycin, suppresses SASP but not cell cycle arrest (Laberge et al., 2015; Wang et al., 2017).
Developmental senescence is characterized by common markers of senescent cells, such as senescence‐associated β‐galactosidase (SA‐β‐Gal), p21, and p15; however, it is not accompanied by DDR and secretion of major SASP factors (Munoz‐Espin et al., 2013; Storer et al., 2013). Thus, in some physiological contexts, senescence develops without an inflammatory response, and SASP in its conventional form (“canonical SASP”) is not mandatory for acquisition of senescence.
The blind mole rat, Spalax, is a long‐lived, cancer‐resistant subterranean mammal that has evolved efficient survival mechanisms and adaptations to stressful conditions in its underground hypoxic and hypercapnic environment (Avivi et al., 2010; Schulke et al., 2012; Shams, Avivi, & Nevo, 2005). Spalax evolved unique capability to maintain oxygen homeostasis by rapid erythrocyte production, which is achieved by an adaptive elevation in Epo mRNA expression and the ability to extract iron from ferritin (Iancu, Arad, Shams, & Manov, 2014; Shams, Nevo, & Avivi, 2005). These and other features that Spalax evolved during 40 million years of evolution underground allow it to cope with continuous stress and maintain strong cellular and tissue homeostasis, apparently providing resistance to cancer and a prolonged lifespan. Recently, we demonstrated that Spalax skin fibroblasts successfully resist genotoxic stress through effective DNA repair, compared with fibroblasts of Rattus (Domankevich, Eddini, Odeh, & Shams, 2018). These data explain, at least in part, Spalax resistance to chemical‐induced carcinogenesis observed earlier (Manov et al., 2013). In this study, we focused on cellular senescence in Spalax fibroblasts. Two models of cellular senescence, replicative senescence (RS) and etoposide‐induced senescence (EIS), were used to evaluate proliferative arrest and senescent secretory phenotype in Spalax primary fibroblasts, in comparison with mouse and human cells. Our data revealed neither substantial DNA damage nor enhanced expression of well‐characterized pro‐inflammatory SASP factors: interleukin‐6 (IL6), IL8, IL1α, growth‐related oncogene alpha (GROα), SerpinB2, intercellular adhesion molecule (ICAM‐1), and cyclooxygenase‐2 (Cox2). Reduced mRNA expression of pro‐inflammatory SASP representatives was also found in aging Spalax tissues. The secretory phenotype of Spalax senescent cells that lack basic inflammatory factors, termed here “noncanonical SASP,” was investigated, and several molecular aspects of its regulation in Spalax were evaluated.
2.1 Spalax fibroblasts, similarly to human and mouse fibroblasts, undergo replicative and etoposide‐induced senescence
Fibroblasts were subjected to serial passages, and senescence phenotype was defined as a state in which most of the cells have acquired an enlarged, flattened morphology, the number of dividing cells significantly decreased, and most cells exhibit positive SA‐β‐Gal. Similar to mouse cells, Spalax fibroblasts became senescent at passages 5–8, while for human fibroblasts, more than 40 passages were required to bring about senescence (the cells completely lost their ability to divide at passage #55–60). Noteworthy, replicative capacity of mammalian fibroblasts in culture does not reflect species longevity but correlates well with body size (Lorenzini, Tresini, Austad, & Cristofalo, 2005). Therefore, human fibroblasts took more divisions to achieve senescence. Late‐passage cells (#6–8 for Spalax; #5‐6 for mice; and above # 46 for human) showed a significant increase in SA‐β‐Gal activity compared with early‐passage ones (Figure S1A,B). The population doubling rates of young fibroblasts of Spalax, mice, and human were the same; cells underwent 3.125 divisions, and the number of cells increased from 2.5 × 104 to 250 × 104 in 96 hr. Thus, the in vitro experimental conditions that we used provided the same proliferative activity for growing human, Spalax, and mouse fibroblasts (Figure S1C). Proliferative capabilities gradually decreased with aging. Once the fibroblasts reached the stage of replicative aging, they ceased to divide, remained alive, and could be subjected to further passages in a 1:1 ratio.
To investigate DNA damage‐induced senescence, fibroblasts were subjected to etoposide, a chemotherapeutic compound that induces DNA damage through the inhibition of DNA topoisomerase II (Yang et al., 2009). Etoposide induces cellular senescence or apoptosis, depending on the concentration used. Spalax and mouse fibroblasts (passage #2–4) and human primary fibroblasts (passage #3–6) were treated with either 1 µg/ml etoposide (Leontieva & Blagosklonny, 2010) or DMSO (control) for 5 days. The cells were then washed and either collected for analysis or replated and incubated for an additional 3 days, followed by SA‐β‐Gal activity staining and EdU (5‐ethynyl‐2‐deoxyuridine) incorporation assay (a detailed scheme of the experiments is presented in Figure 1a). The number of SA‐β‐Gal‐positive cells was significantly increased in EIS fibroblasts from Spalax, mice, and human, compared with the control (Figure 1b,c). Spalax fibroblasts exposed to etoposide exhibited an irreversible growth arrest, confirmed by decreased EdU incorporation following low‐density replating and incubation for an additional 3 days (in the absence of etoposide; Figure 1d). The percentages of EdU‐positive nuclei (representing proliferating cells) were 35.65 ± 5.17% and 31.09 ± 8.18% among untreated human and Spalax cells, respectively (Figure 1e). Among etoposide‐treated human cells, only 14.21 ± 10.5% EdU‐positive nuclei were observed (p ≤ .01 vs. control), whereas no EdU‐positive nuclei were detected among EIS Spalax fibroblasts. Flow cytometry has shown that etoposide‐induced proliferative arrest was due to an accumulation of cells in the S‐G2/M phases of the cell cycle (Figure 1f).
Figure 1
Etoposide‐induced senescence (EIS). (a) Schematic illustration of the experimental design. (b) SA‐β‐Gal staining representative images. Fibroblasts were treated with DMSO (control) or with 1 μg/ml etoposide for 5 days, washed, and incubated for an additional 3 days without etoposide. © Percentage of SA‐β‐Gal‐positive cells calculated from at least 300 cells in four independent fields for each biological repeat (n = 3) in triplicate (Spalax and mouse cells were isolated from three independent individuals). The mean value of each species is compared to its control cells (DMSO‐treated), *** p < .001. (d) Representative fluorescence images demonstrating the levels of EdU incorporation in Spalax and human untreated and etoposide‐treated cells. (e) Quantitative data showing the % of EdU‐positive nuclei in untreated and etoposide‐treated Spalax and human fibroblasts. Experiments were repeated two times (n = 2) in triplicate. (f) Flow cytometry analysis showing the percentage of cells in cell cycle stages. Values represent the averages of at least two independent experiments in triplicate. EdU, 5‐ethynyl‐2‐deoxyuridine

Also tagged with one or more of these keywords: cellular senescence, dna damage, dna repair, interleukin‐1 alpha (il1α), nuclear factor κb (nf‐κb), senescence‐associated secretory phenotype (sasp), spalax

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