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The ELN gene encodes tropoelastin which is used to generate elastic fibers that insure proper tissue elasticity. Decreased amounts of elastic fibers and/or accumulation of bioactive products of their cleavage, named elastokines, are thought to contribute to aging. Cellular senescence, characterized by a stable proliferation arrest and by the senescence-associated secretory phenotype (SASP), increases with aging, fostering the onset and progression of age-related diseases and overall aging, and has so far never been linked with elastin. Here, we identified that decrease in ELN either by siRNA in normal human fibroblasts or by knockout in mouse embryonic fibroblasts results in premature senescence. Surprisingly this effect is independent of elastic fiber degradation or elastokines production, but it relies on the rapid increase in HMOX1 after ELN downregulation. Moreover, the induction of HMOX1 depends on p53 and NRF2 transcription factors, and leads to an increase in iron, further mediating ELN downregulation-induced senescence. Screening of iron-dependent DNA and histones demethylases revealed a role for histone PHF8 demethylase in mediating ELN downregulation-induced senescence. Collectively, these results unveil a role for ELN in protecting cells from cellular senescence through a non-canonical mechanism involving a ROS/HMOX1/iron accumulation/PHF8 histone demethylase pathway reprogramming gene expression towards a senescence program.

Abstract Background TGFβ induces several cell phenotypes including senescence, a stable cell cycle arrest accompanied by a secretory program, and epithelial-mesenchymal transition (EMT) in normal epithelial cells. During carcinogenesis cells lose the ability to undergo senescence in response to TGFβ but they maintain an EMT, which can contribute to tumor progression. Our aim was to identify mechanisms promoting TGFβ-induced senescence escape. Methods In vitro experiments were performed with primary human mammary epithelial cells (HMEC) immortalized by hTert. For kinase library screen and modulation of gene expression retroviral transduction was used. To characterize gene expression, RNA microarray with GSEA analysis and RT-qPCR were used. For protein level and localization, Western blot and immunofluorescence were performed. For senescence characterization crystal violet assay, Senescence Associated-β-Galactosidase activity, EdU staining were conducted. To determine RSK3 partners FLAG-baited immunoprecipitation and mass spectrometry-based proteomic analyses were performed. Proteosome activity and proteasome enrichment assays were performed. To validate the role of RSK3 in human breast cancer, analysis of METABRIC database was performed. Murine intraductal xenografts using MCF10DCIS.com cells were carried out, with histological and immunofluorescence analysis of mouse tissue sections. Results A screen with active kinases in HMECs upon TGFβ treatment identified that the serine threonine kinase RSK3, or RPS6KA2, a kinase mainly known to regulate cancer cell death including in breast cancer, reverted TGFβ-induced senescence. Interestingly, RSK3 expression decreased in response to TGFβ in a SMAD3-dependent manner, and its constitutive expression rescued SMAD3-induced senescence, indicating that a decrease in RSK3 itself contributes to TGFβ-induced senescence. Using transcriptomic analyses and affinity purification coupled to mass spectrometry-based proteomics, we unveiled that RSK3 regulates senescence by inhibiting the NF-κΒ pathway through the decrease in proteasome-mediated IκBα degradation. Strikingly, senescent TGFβ-treated HMECs display features of epithelial to mesenchymal transition (EMT) and during RSK3-induced senescence escaped HMECs conserve EMT features. Importantly, RSK3 expression is correlated with EMT and invasion, and inversely correlated with senescence and NF-κΒ in human claudin-low breast tumors and its expression enhances the formation of breast invasive tumors in the mouse mammary gland. Conclusions We conclude that RSK3 switches cell fate from senescence to malignancy in response to TGFβ signaling.

Abstract Although aging is a major risk factor for most types of cancers, it is barely studied in this context. The transmembrane protein PLA2R1 (phospholipase A2 receptor) promotes cellular senescence, which can inhibit oncogene-induced tumor initiation. Functions and mechanisms of action of PLA2R1 during aging are largely unknown. In this study, we observed that old Pla2r1 knockout mice were more prone to spontaneously develop a wide spectrum of tumors compared to control littermates. Consistently, these knockout mice displayed increased Parp1, a master regulator of DNA damage repair, and decreased DNA damage, correlating with large human dataset analysis. Forced PLA2R1 expression in normal human cells decreased PARP1 expression, induced DNA damage and subsequent senescence, while the constitutive expression of PARP1 rescued cells from these PLA2R1-induced effects. Mechanistically, PARP1 expression is repressed by a ROS (reactive oxygen species)-Rb-dependent mechanism upon PLA2R1 expression. In conclusion, our results suggest that PLA2R1 suppresses aging-induced tumors by repressing PARP1, via a ROS–Rb signaling axis, and inducing DNA damage and its tumor suppressive responses.

Contacts between mitochondria and endoplasmatic reticulum (ER), and the transfer of calcium between them, have an important role in the regulation of cellular phenotypes, including senescence. Here the authors show that ITPR2 deficient mice display improved aging, associated with a decreased number of contacts between the mitochondria and the ER.

In response to many stresses, such as oncogene activation or DNA damage, cells can enter cellular senescence, a state of proliferation arrest accompanied by a senescence-associated secretory phenotype (SASP). Cellular senescence plays a key role in many physiopathological contexts, including cancer, aging and aging-associated diseases, therefore, it is critical to understand how senescence is regulated. Calcium ions (Ca2+) recently emerged as pivotal regulators of cellular senescence. However, how Ca2+ levels are controlled during this process is barely known. Here, we report that intracellular Ca2+ contents increase in response to many senescence inducers in immortalized human mammary epithelial cells (HMECs) and that expression of calbindin 1 (CALB1), a Ca2+-binding protein, is upregulated in this context, through the Ca2+-dependent calcineurin/NFAT pathway. We further show that overexpression of CALB1 buffers the rise in intracellular Ca2+ levels observed in senescent cells. Finally, we suggest that increased expression of Ca2+-binding proteins calbindins is a frequent mark of senescent cells. This work thus supports that, together with Ca2+channels, Ca2+-binding proteins modulate Ca2+ levels and flux during cellular senescence. This opens potential avenues of research to better understand the role of Ca2+ and of Ca2+-binding proteins in regulating cellular senescence.

Staphylococcus lugdunensis is a coagulase negative Staphylococcus recognized as a virulent pathogen. It is responsible for a wide variety of infections, some of which are associated with biofilm production, such as implanted medical device infections or endocarditis. However, little is known about S. lugdunensis regulation of virulence factor expression. Two-component regulatory systems (TCS) play a critical role in bacterial adaptation, survival, and virulence. Among them, LytSR is widely conserved but has variable roles in different organisms, all connected to metabolism or cell death and lysis occurring during biofilm development. Therefore, we investigated here the functions of LytSR in S. lugdunensis pathogenesis. Deletion of lytSR in S. lugdunensis DSM 4804 strain did not alter either susceptibility to Triton X-100 induced autolysis or death induced by antibiotics targeting cell wall synthesis. Interestingly, ΔlytSR biofilm was characterized by a lower biomass, a lack of tower structures, and a higher rate of dead cells compared to the wild-type strain. Virulence toward Caenorhabditis elegans using a slow-killing assay was significantly reduced for the mutant compared to the wild-type strain. By contrast, the deletion of lytSR had no effect on the cytotoxicity of S. lugdunensis toward the human keratinocyte cell line HaCaT. Transcriptional analyses conducted at mid- and late-exponential phases showed that lytSR deletion affected the expression of 286 genes. Most of them were involved in basic functions such as the metabolism of amino acids, carbohydrates, and nucleotides. Furthermore, LytSR appeared to be involved in the regulation of genes encoding known or putative virulence and colonization factors, including the fibrinogen-binding protein Fbl, the major autolysin AtlL, and the type VII secretion system. Overall, our data suggest that the LytSR TCS is implicated in S. lugdunensis pathogenesis, through its involvement in biofilm formation and potentially by the control of genes encoding putative virulence factors.

Genetic alterations have recently been described as emerging during the culture of embryonic stem cells or induced pluripotent stem cells, raising concerns about their safety in future clinical use. Myoblasts are adult stem cells with important therapeutic potential that have been used in clinical trials for almost 20 years, but their genome integrity has not yet been established. Here we produced 10 human myoblast preparations and investigated their genomic stability. At the third passage, half of the preparations had a normal karyotype and half showed one to four alterations/30 metaphases. Chromosome 2 trisomy was found in 1–2/30 meta-phases and/or 2/100 nuclei by FISH in 3/10 samples, and there was no other recurrent anomaly. When prolonging cultures, these erratic abnormalities were never associated with a growth advantage. Cellular senescence was manifested in all samples by growth arrest before passage 15. Expression of TERT was always negative. Molecular analysis of individual p53 transcripts did not reveal tumorigenic mutations. CGH array (10 samples) and exome sequencing (one sample) failed to detect copy number variations or accumulation of mutations, respectively. Myoblasts did not grow either in soft agar or in vivo after injection in immunodeficient mice. Hence, occasional genomic abnormalities may occur during myoblast culture but are not associated with risk of transformation.