Senescence In Mouse Embryonic Fibroblasts Research Articles

TP73 Isoform-specific disruption reveals a critical role of TAp73beta in growth suppression and inflammatory response

TP73 is expressed as multiple N- and C-terminal isoforms through two separate promoters or alternative splicing. While N-terminal p73 isoforms have been well studied, very little is known about p73 C-terminal isoforms. Thus, CRISPR was used to delete TP73 Exon13 (E13-KO) to induce p73α to p73β isoform switch. We showed that E13-KO led to decreased cell proliferation and migration and sensitized cells to ferroptosis, which can be reverted by knockdown of TAp73β in E13-KO cells. To understand the biological function of p73β in vivo, we generated a mouse model in that the Trp73 E13 was deleted by CRISPR. We showed that p73α to p73β isoform switch led to increased cellular senescence in mouse embryonic fibroblasts. We also showed that E13-deficient mice exhibited shorter life span and were prone to spontaneous tumors, chronic inflammation and liver steatosis as compared to WT mice. Additionally, we found that the incidence of chronic inflammation and liver steatosis was higher in E13-deficient mice than that in Trp73-deficient mice, suggesting that p73β is a strong inducer of inflammatory response. Mechanistically, we showed that TAp73β was able to induce cysteine dioxygenase 1 (CDO-1), leading to cysteine depletion and subsequently, enhanced ferroptosis and growth suppression. Conversely, knockdown of CDO-1 was able to alleviate the growth suppression and ferroptosis in E13-KO cells. Together, our data suggest that at a physiologically relevant level, TAp73β is a strong inducer of growth suppression but insufficient to compensate for loss of TAp73α in tumor suppression due to aberrant induction of inflammatory response and liver steatosis.

Senescent cells limit p53 activity via multiple mechanisms to remain viable

Super-enhancers regulate genes with important functions in processes that are cell type-specific or define cell identity. Mouse embryonic fibroblasts establish 40 senescence-associated super-enhancers regardless of how they become senescent, with 50 activated genes located in the vicinity of these enhancers. Here we show, through gene knockdown and analysis of three core biological properties of senescent cells that a relatively large number of senescence-associated super-enhancer-regulated genes promote survival of senescent mouse embryonic fibroblasts. Of these, Mdm2, Rnase4, and Ang act by suppressing p53-mediated apoptosis through various mechanisms that are also engaged in response to DNA damage. MDM2 and RNASE4 transcription is also elevated in human senescent fibroblasts to restrain p53 and promote survival. These insights identify key survival mechanisms of senescent cells and provide molecular entry points for the development of targeted therapeutics that eliminate senescent cells at sites of pathology.

P73α1, a p73 C-terminal isoform, regulates tumor suppression and the inflammatory response via Notch1

p73, a p53 family member, undergoes alternative splicing at the 3′ end to produce multiple isoforms, but their expression and activity are largely unknown. Thus, CRISPR was used to knock out exon 12 (E12) in human cancer cell lines and mice, leading to isoform switch from p73α to isoform p73α1. We found that p73α1 is naturally expressed and induced by DNA damage. We also found that knockout of E12 suppresses cell growth and migration in H1299 and MIA PaCa-2 cells and promotes cellular senescence in mouse embryonic fibroblasts. Similarly, ectopic expression of p73α1 suppresses cell proliferation, whereas knockdown of p73α1 restores the cell proliferative and migratory capacities of E12−/− cells. Consistently, we found that E12+/− mice are not prone to spontaneous tumors. Instead, E12+/− mice are prone to systemic inflammation and exhibit elevated TNFα expression in inflamed tissues. Moreover, we found that Notch1, a master regulator of the inflammatory response, is regulated by p73α1 and highly expressed in E12−/− cells and inflamed E12+/− mouse tissues. Furthermore, through knockdown of p73α1 and/or Notch1 in E12−/− cells, we found that Notch1 is necessary for p73α1-mediated growth suppression. Together, these data suggest that p73α1 plays a critical role in tumor suppression and the inflammatory response via Notch1.

Circular RNA circRNF169 functions as a miR-30c-5p sponge to promote cellular senescence

Circular RNAs (circRNAs), characterized as single-stranded closed circular RNA molecules, have been established to exert pivotal functions in various biological or pathological processes. Nonetheless, the effects and underlying mechanisms concerning circRNAs on the aging and aging-related diseases remain elusive. We herein compared the expression patterns of circRNAs in young and senescent mouse embryonic fibroblasts (MEFs), and uncovered that circRNF169 was dramatically up-regulated in senescent MEFs compared with that in young MEFs. Therefore, we further digged into the role and potential mechanisms of circRNF169 in the senescence of MEFs. The results of senescence-associate-β-galactosidase staining and BrdU incorporation assay showed that silencing of circRNF169 significantly delayed MEFs senescence and promoted cell proliferation, while ectopic expression of circRNF169 exhibited the opposite effects. Moreover, the dual-luciferase reporter assay confirmed that circRNF169 acted as an endogenous miR-30c-5p sponge, which accelerated cellular senescence by sequestering and inhibiting miR-30c-5p activity. Taken together, our results suggested that circRNF169 exerted a crucial role in cellular senescence through sponging miR-30c-5p and represented a promising target for aging intervention.

Bioorthogonal Conjugation-Assisted Purification Method for Profiling Cell Surface Proteome.

Surface biotinylation has been widely adapted in profiling the cellular proteome associated with the plasma membrane. However, the workflow is subject to interference from the cytoplasmic biotin-associated proteins that compete for streptavidin-binding during purification. Here we established a bioorthogonal conjugation-assisted purification (BCAP) workflow that utilizes the Staudinger chemoselective ligation to label and isolate surface-associated proteins while minimizing the binding of endogenous biotin-associated proteins. Label-free quantitative proteomics demonstrated that BCAP is efficient in isolating cell surface proteins with excellent reproducibility. Subsequently, we applied BCAP to compare the surface proteome of proliferating and senescent mouse embryonic fibroblasts (MEFs). Among the results, EHD2 was identified and validated as a novel protein that is enhanced at the cell surface of senescent MEFs. We expect that BCAP will have broad applications in profiling cell surface proteomes in the future.

Novel small molecule inhibition of IKK/NF-κB activation reduces markers of senescence and improves healthspan in mouse models of aging.

Constitutive NF‐κB activation is associated with cellular senescence and stem cell dysfunction and rare variants in NF‐κB family members are enriched in centenarians. We recently identified a novel small molecule (SR12343) that inhibits IKK/NF‐κB activation by disrupting the association between IKKβ and NEMO. Here we investigated the therapeutic effects of SR12343 on senescence and aging in three different mouse models. SR12343 reduced senescence‐associated beta‐galactosidase (SA‐β‐gal) activity in oxidative stress‐induced senescent mouse embryonic fibroblasts as well as in etoposide‐induced senescent human IMR90 cells. Chronic administration of SR12343 to the Ercc1 −/ ∆ and Zmpste24 −/− mouse models of accelerated aging reduced markers of cellular senescence and SASP and improved multiple parameters of aging. SR12343 also reduced markers of senescence and increased muscle fiber size in 2‐year‐old WT mice. Taken together, these results demonstrate that IKK/NF‐κB signaling pathway represents a promising target for reducing markers of cellular senescence, extending healthspan and treating age‐related diseases.

Bifidobacterium adolescentis regulates catalase activity and host metabolism and improves healthspan and lifespan in multiple species.

To identify candidate bacteria associated with aging, we performed fecal microbiota sequencing in young, middle-aged and older adults, and found lower Bifidobacterium adolescentis abundance in older individuals aged ≥60 years. Dietary supplementation of B. adolescentis improved osteoporosis and neurodegeneration in a mouse model of premature aging (Terc-/-) and increased healthspan and lifespan in Drosophila melanogaster and Caenorhabditis elegans. B. adolescentis supplementation increased the activity of the catalase (CAT) enzyme in skeletal muscle and brain tissue from Terc-/- mice, and suppressed cellular senescence in mouse embryonic fibroblasts. Transgenic deletion of catalase (ctl-2) in C. elegans abolished the effects of B. adolescentis on the lifespan and healthspan. B. adolescentis feeding also led to changes in oxidative stress-associated metabolites in Terc-/- mouse feces. These results suggest a role for B. adolescentis in improving the healthspan and lifespan through the regulation of CAT activity and host metabolism.

Cytosolic dsDNA is a novel senescence marker associated with pyroptosis activation

Cellular senescence has become a research focus because of its dual roles in ageing and tumorigenesis. The biomarkers of senescence are essential for detecting senescent cells and understanding the ageing process and its regulation. Here, we identify cytosolic double-stranded DNA (dsDNA) as a novel sensitive biomarker for cellular senescence of mouse embryonic fibroblasts (MEFs) in response to common types of stimuli, including replicative stress, genetic modification and oxidative stress. We found that the accumulation of cytosolic dsDNA was positively correlated with the senescence process in MEFs and was detectable earlier than senescence-associated β-galactosidase (SA-β-Gal) staining, which is the current gold standard for senescence detection. Due to the immunogenicity of dsDNA, we further investigated the stimulation of two dsDNA sensors, cyclic guanosine monophosphate (GMP)–adenosine monophosphate (AMP) (cGAMP) synthase (cGAS) and absent in melanoma-2 (AIM2). The results showed that the cGAS protein level did not significantly change upon senescence stimulation, while AIM2 expression was significantly upregulated in senescent cells. Surprisingly, we found that ageing-related cytosolic dsDNA induced significant pyroptosis activation in the senescent MEFs. These data reveal novel easy-to-detect biomarker for cellular senescence. The activation of downstream immunological response pathways might add new experimental evidence for inflammatory ageing.

Identification of GL-V9 as a novel senolytic agent against senescent breast cancer cells

Senescent cancer cells contribute to tumor refractoriness. The removal of senescent cells after chemotherapy prevents or delays cancer relapse. Our study showed that GL-V9 (5-hydroxy-8-methoxy-2-phenyl-7-(4-(pyrrolidin-1-yl) butoxy)-4-H-chromen-4-one), a potential anticancer drug, eliminated senescent MEFs (Mouse embryonic fibroblasts) and drug-induced senescent breast cancer cells. GL-V9 induced apoptosis in senescent MDA-MB-231 cells. Mechanistically, it alkalized lysosomes and increased the abundance of mitochondria as well as ROS (Reactive oxygen species). The senolytic effect of GL-V9 was also observed in epirubicin-treated mammary tumors in MMTV-PyMT mice. Our data thus indicated that GL-V9 is a promising senolytic drug which could be used to improve the outcome of cancer chemotherapy.

Characterization of HMGA1P6 transgenic mouse embryonic fibroblasts

ABSTRACT Latest studies have shown that deregulated pseudogene transcripts contribute to cancer working as competing endogenous RNAs. Our research group has recently demonstrated that the overexpression of two HMGA1 pseudogenes, HMGA1P6 and HMGA1P7, has a critical role in cancer progression. These pseudogenes work sustaining the expression of HMGA1 and other cancer-related genes. We generated a mouse model overexpressing HMGA1P6 to better study the HMGA1-pseudogene function in a more physiological context. Here, we show the proliferation rate and the susceptibility to senescence of mouse embryonic fibroblasts obtained from HMGA1P6-overexpressing mice to better characterize the HMGA1-pseudogene function. Indeed, our study reports that mouse embryonic fibroblasts (MEFs) derived from HMGA1P6 mice express higher HMGA1 mRNA and protein levels. Moreover, these cells grow faster and senesce later than wild-type sustaining the oncogenic role of ceRNA crosstalk mediated by HMGA1Ps.

Single-Cell Transcriptome Analysis Reveals Six Subpopulations Reflecting Distinct Cellular Fates in Senescent Mouse Embryonic Fibroblasts.

Replicative senescence is a hallmark of aging, which also contributes to individual aging. Mouse embryonic fibroblasts (MEFs) provide a convenient replicative senescence model. However, the heterogeneity of single MEFs during cellular senescence has remained unclear. Here, we conducted single-cell RNA sequencing on senescent MEFs. Principal component analysis showed obvious heterogeneity among these MEFs such that they could be divided into six subpopulations. Three types of gene expression analysis revealed distinct expression features of these six subpopulations. Trajectory analysis revealed three distinct lineages during MEF senescence. In the main lineage, some senescence-associated secretory phenotypes were upregulated in a subset of cells from senescent clusters, which could not be distinguished in a previous bulk study. In the other two lineages, a possibility of escape from cell cycle arrest and coupling between translation-related genes and ATP synthesis-related genes were also discovered. Additionally, we found co-expression of transcription factor HOXD8 coding gene and its potential target genes in the main lineage. Overexpression of Hoxd8 led to senescence-associated phenotypes, suggesting HOXD8 is a new regulator of MEF senescence. Together, our single-cell sequencing on senescent MEFs largely expanded the knowledge of a basic cell model for aging research.

Down-Regulation of Fibroblast Growth Factor 2 (FGF2) Contributes to the Premature Senescence of Mouse Embryonic Fibroblast.

BackgroundFreshly isolated mouse embryonic fibroblasts (MEFs) have great proliferation capacity but quickly enter senescent state after several rounds of cell cycle, a process called premature senescence. Cellular senescence can be induced by various stresses such as telomere erosion, DNA damage, and oncogenic signaling. But the contribution of other molecules, such as growth factors, to cellular senescence is incompletely understood. This study aimed to compare the gene expression difference between non-senescent and senescent MEFs to identify the key molecule(s) involved in the spontaneous senescence of MEFs.Material/MethodsPrimary MEFs were isolated from E12.5 pregnant C57/BL6 mice. The cells were continuously cultured in Dulbecco’s Modified Eagle Medium for 9 passages. SA-β-Gal staining was used as an indicator of cell senescence. The supernatant from primary MEFs (P1 medium) or Passage 6 MEFs (P6 medium) were used to culture freshly isolated MEFs to observe the effects on cell senescence state. Gene expression profiles of primary and senescent MEFs were investigated by RNA-Seq to find the key genes involved in cell senescence. Adipocyte differentiation assay was used to evaluate the stemness of MEFs cultured in FGF2-stimulated medium.ResultsThe senescence of MEFs cultured in the P1 medium was alleviated when compared to the P6 medium. Downregulation of FGF2 expression was revealed by RNA-Seq and further confirmed by real-time quantitative polymerase chain reaction and western blot. FGF2-stimulated medium also had anti-senescence function and could maintain the differentiation ability of MEFs.ConclusionsThe premature senescence of MEFs was at least partially caused by FGF2 deficiency. Exogenous FGF2 could alleviate the senescent phenotype.

MTORC1-Rps15 Axis Contributes to the Mechanisms Underlying Global Translation Reduction During Senescence of Mouse Embryonic Fibroblasts.

The reduction of protein translation is a common feature in senescent cells and aging organisms, yet the underlying mechanisms are not fully understood. Here we show that both global mRNA translation and mammalian/mechanistic target of rapamycin complex 1 (mTORC1) kinase activity are declined in a senescent model of mouse embryonic fibroblasts (MEFs). Furthermore, RNA-seq analyses from polysomal versus total mRNA fractions identify TOP-like mRNA of Rps15 whose translation is regulated by mTORC1 during MEF senescence. Overexpression of Rps15 delays MEF senescence, possibly through regulating ribosome maturation. Together, these findings indicate that the activation of mTORC1-Rps15 axis ameliorate senescence by regulating ribosome biogenesis, which may provide further insights into aging research.

Cortical neurons develop a senescence-like phenotype promoted by dysfunctional autophagy.

Senescent cells accumulate in various tissues and organs with aging altering surrounding tissue due to an active secretome, and at least in mice their elimination extends healthy lifespan and ameliorates several chronic diseases. Whether all cell types senesce, including post-mitotic cells, has been poorly described mainly because cellular senescence was defined as a permanent cell cycle arrest. Nevertheless, neurons with features of senescence have been described in old rodent and human brains. In this study we characterized an in vitro model useful to study the molecular basis of senescence of primary rat cortical cells that recapitulates senescent features described in brain aging. We found that in long-term cultures, rat primary cortical neurons displayed features of cellular senescence before glial cells did, and developed a functional senescence-associated secretory phenotype able to induce paracrine premature senescence of mouse embryonic fibroblasts but proliferation of rat glial cells. Functional autophagy seems to prevent neuronal senescence, as we observed an autophagic flux reduction in senescent neurons both in vitro and in vivo, and autophagy impairment induced cortical cell senescence while autophagy stimulation inhibited it. Our findings suggest that aging-associated dysfunctional autophagy contributes to senescence transition also in neuronal cells.

Pak2 kinase promotes cellular senescence and organismal aging

Cellular senescence defines an irreversible cell growth arrest state linked to loss of tissue function and aging in mammals. This transition from proliferation to senescence is typically characterized by increased expression of the cell-cycle inhibitor p16INK4a and formation of senescence-associated heterochromatin foci (SAHF). SAHF formation depends on HIRA-mediated nucleosome assembly of histone H3.3, which is regulated by the serine/threonine protein kinase Pak2. However, it is unknown if Pak2 contributes to cellular senescence. Here, we show that depletion of Pak2 delayed oncogene-induced senescence in IMR90 human fibroblasts and oxidative stress-induced senescence of mouse embryonic fibroblasts (MEFs), whereas overexpression of Pak2 accelerated senescence of IMR90 cells. Importantly, depletion of Pak2 in BubR1 progeroid mice attenuated the onset of aging-associated phenotypes and extended life span. Pak2 is required for expression of genes involved in cellular senescence and regulated the deposition of newly synthesized H3.3 onto chromatin in senescent cells. Together, our results demonstrate that Pak2 is an important regulator of cellular senescence and organismal aging, in part through the regulation of gene expression and H3.3 nucleosome assembly.

Real-Time Imaging of Mitochondrial ATP Dynamics Reveals the Metabolic Setting of Single Cells

SummaryReprogramming of metabolic pathways determines cell functions and fate. In our work, we have used organelle-targeted ATP biosensors to evaluate cellular metabolic settings with high resolution in real time. Our data indicate that mitochondria dynamically supply ATP for glucose phosphorylation in a variety of cancer cell types. This hexokinase-dependent process seems to be reversed upon the removal of glucose or other hexose sugars. Our data further verify that mitochondria in cancer cells have increased ATP consumption. Similar subcellular ATP fluxes occurred in young mouse embryonic fibroblasts (MEFs). However, pancreatic beta cells, senescent MEFs, and MEFs lacking mitofusin 2 displayed completely different mitochondrial ATP dynamics, indicative of increased oxidative phosphorylation. Our findings add perspective to the variability of the cellular bioenergetics and demonstrate that live cell imaging of mitochondrial ATP dynamics is a powerful tool to evaluate metabolic flexibility and heterogeneity at a single-cell level.

Time-dependent modulation of FoxO activity by HDAC inhibitor in oncogene-transformed E1A+Ras cells.

HDAC inhibitors (HDACIs) induce irreversible cell cycle arrest and senescence in mouse embryonic fibroblasts transformed with E1A and c-Ha-Ras oncogenes (E1A+Ras cell line). The aging rate has been associated with the production of high levels of Reactive Oxygen Species (ROS). Specific increases of ROS level have been demonstrated as potentially critical for induction and maintenance of cell senescence process. It's known that HDACs regulate the ROS-dependent FoxO factors, which are responsible for cell growth, proliferation, and longevity. The characteristic ROS increase during aging may be responsible for the decreased HDAC activity, which facilitates the senescent-like phenotype. The objective of this study was to investigate the impact of FoxO transcription factors on HDACIs-induced senescence of E1A+Ras oncogenes transformed cells. This study shows the specific time-dependent effect of HDACI sodium butyrate treatment on FoxO proteins in E1A+Ras cells. Indeed, short-term treatment with NaB results in FoxO activation, which takes place through nuclear translocation, and accompanied by accumulation of such ROS scavengers as MnSOD and SOD2. However, prolonged treatment leads to extensive FoxO degradation and increased intracellular levels of ROS. This degradation is connected with NaB-induced activation of Akt kinase. All of these findings establish that one of the possible mechanism involved in NaB-induced senescence of transformed cells is mediated through down-regulation of FoxO transcription factors and ROS accumulation.

Copper accumulation in senescent cells: Interplay between copper transporters and impaired autophagy

Cellular senescence is characterized by irreversible growth arrest incurred through either replicative exhaustion or by pro-oncogenic cellular stressors (radioactivity, oxidative stress, oncogenic activation). The enrichment of senescent cells in tissues with age has been associated with tissue dyshomeostasis and age-related pathologies including cancers, neurodegenerative disorders (e.g. Alzheimer's, Parkinson's, etc.) and metabolic disorders (e.g. diabetes). We identified copper accumulation as being a universal feature of senescent cells [mouse embryonic fibroblasts (MEF), human prostate epithelial cells and human diploid fibroblasts] in vitro. Elevated copper in senescent MEFs was accompanied by elevated levels of high-affinity copper uptake protein 1 (Ctr1), diminished levels of copper-transporting ATPase 1 (Atp7a) (copper export) and enhanced antioxidant defence reflected by elevated levels of glutathione (GSH), superoxide dismutase 1 (SOD1) and glutaredoxin 1 (Grx1). The levels of intracellular copper were further increased in senescent MEFs cultured in copper supplemented medium and in senescent Mottled Brindled (Mobr) MEFs lacking functional Atp7a. Finally, we demonstrated that the restoration/preservation of autophagic-lysosomal degradation in senescent MEFs following rapamycin treatment correlated with attenuation of copper accumulation in these cells despite a further decrease in Atp7a levels. This study for the first time establishes a link between Atp7a and the autophagic-lysosomal pathway, and a requirement for both to effect efficient copper export. Such a connection between cellular autophagy and copper homeostasis is significant, as both have emerged as important facets of age-associated degenerative disease.

Caveolin-1 promotes the tumor suppressor properties of oncogene-induced cellular senescence

Oncogene-induced senescence (OIS) is considered a powerful tumor suppressor mechanism. Caveolin-1 acts as a scaffolding protein to functionally regulate signaling molecules. We demonstrate that a lack of caveolin-1 expression inhibits oncogenic K-Ras (K-RasG12V)-induced premature senescence in mouse embryonic fibroblasts and normal human bronchial epithelial cells. Oncogenic K-Ras induces senescence by limiting the detoxification function of MTH1. We found that K-RasG12V promotes the interaction of caveolin-1 with MTH1, which results in inhibition of MTH1 activity. Lung cancer cells expressing oncogenic K-Ras have bypassed the senescence barrier. Interestingly, overexpression of caveolin-1 restores cellular senescence in both A549 and H460 lung cancer cells and inhibits their transformed phenotype. In support of these findings, our in vivo data demonstrate that overexpression of oncogenic K-Ras (K-RasG12D) induces cellular senescence in the lung of wildtype but not caveolin-1-null mice. A lack of K-RasG12D-induced premature senescence in caveolin-1-null mice results in the formation of more abundant lung tumors. Consistent with these data, caveolin-1-null mice overexpressing K-RasG12D display accelerated mortality. Finally, our animal data were supported by human sample analysis in which we show that caveolin-1 expression is dramatically down-regulated in lung adenocarcinomas from lung cancer patients, both at the mRNA and protein levels, and that low caveolin-1 expression is associated with poor survival. Together, our data suggest that lung cancer cells escape oncogene-induced premature senescence through down-regulation of caveolin-1 expression to progress from premalignant lesions to cancer.

H-Ras and K-Ras Oncoproteins Induce Different Tumor Spectra When Driven by the Same Regulatory Sequences.

Genetic studies in mice have provided evidence that H-Ras and K-Ras proteins are bioequivalent. However, human tumors display marked differences in the association of RAS oncogenes with tumor type. Thus, to further assess the bioequivalence of oncogenic H-Ras and K-Ras, we replaced the coding region of the murine K-Ras locus with H-RasG12V oncogene sequences. Germline expression of H-RasG12V or K-RasG12V from the K-Ras locus resulted in embryonic lethality. However, expression of these genes in adult mice led to different tumor phenotypes. Whereas H-RasG12V elicited papillomas and hematopoietic tumors, K-RasG12V induced lung tumors and gastric lesions. Pulmonary expression of H-RasG12V created a senescence-like state caused by excessive MAPK signaling. Likewise, H-RasG12V but not K-RasG12V induced senescence in mouse embryonic fibroblasts. Label-free quantitative analysis revealed that minor differences in H-RasG12V expression levels led to drastically different biological outputs, suggesting that subtle differences in MAPK signaling confer nonequivalent functions that influence tumor spectra induced by RAS oncoproteins. Cancer Res; 77(3); 707-18. ©2016 AACR.