CPEB regulation of human cellular senescence, energy metabolism, and p53 mRNA translation

Cytoplasmic polyadenylation element-binding protein (CPEB) stimulates polyadenylation and translation in germ cells and neurons. Here, we show that CPEB-regulated translation is essential for the senescence of human diploid fibroblasts. Knockdown of CPEB causes skin and lung cells to bypass the M1 crisis stage of senescence; reintroduction of CPEB into the knockdown cells restores a senescence-like phenotype. Knockdown cells that have bypassed senescence undergo little telomere erosion. Surprisingly, knockdown of exogenous CPEB that induced a senescence-like phenotype results in the resumption of cell growth. CPEB knockdown cells have fewer mitochondria than wild-type cells and resemble transformed cells by having reduced respiration and reactive oxygen species (ROS), normal ATP levels, and enhanced rates of glycolysis. p53 mRNA contains cytoplasmic polyadenylation elements in its 3′ untranslated region (UTR), which promote polyadenylation. In CPEB knockdown cells, p53 mRNA has an abnormally short poly(A) tail and a reduced translational efficiency, resulting in an ~50% decrease in p53 protein levels. An shRNA-directed reduction in p53 protein by about 50% also results in extended cellular life span, reduced respiration and ROS, and increased glycolysis. Together, these results suggest that CPEB controls senescence and bioenergetics in human cells at least in part by modulating p53 mRNA polyadenylation-induced translation.     

Metabolic changes during cellular senescence investigated by proton NMR-spectroscopy

Cellular senescence is of growing interest due to its role in tumour suppression and its contribution to organismic ageing. This cellular state can be reached by replicative loss of telomeres or certain stresses in cell culture and is characterized by the termination of cell division; however, the cells remain metabolically active. To identify metabolites that are characteristic for senescent cells, extracts of human embryonic lung fibroblast (WI-38 cell line) have been investigated with NMR spectroscopy. Three different types of senescence have been characterized: replicative senescence, DNA damage-induced senescence (etoposide treatment) and oncogene-induced senescence (hyperactive RAF kinase). The metabolite pattern allows (I) discrimination of senescent and control cells and (II) discrimination of the three senescence types. Senescent cells show an increased ratio of glycerophosphocholine to phosphocholine independent from the type of senescence. The increase in glycerophosphocholine implicates a key role of phospholipid metabolism in cellular senescence. The observed changes in the choline metabolism are diametrically opposite to the well-known changes in choline metabolism of tumour cells. As tumours responding to chemotherapeutic agents show a “glycerophosphocholine-to-phosphocholine switch” i.e. an increase in glycerophosphocholine, our metabolic data suggests that these malignant cells enter a senescent state emphasizing the role of senescence in tumour suppression.     

高脂饮食引起大鼠动脉血管内皮细胞衰老

目的：研究高脂饮食对大鼠血管内皮细胞衰老的影响。方法：大鼠高脂饲料喂养12周后测定血脂的变化,HE染色分析胸主动脉和肾动脉血管结构的改变;胶原酶灌注方法分离胸主动脉和肾动脉血管内皮细胞,应用免疫细胞化学的方法鉴定内皮细胞;应用细胞化学染色的方法检测衰老相关的半乳糖甘酶（senescence-associated β-galactosidase,SA-β-gal）活性。 结果：SD 雄性大鼠高脂饲料喂养12周后,大鼠血浆三酰甘油、总胆固醇、高密度脂蛋白胆固醇水平明显升高, 胸主动脉和肾动脉出现明显的粥样硬化改变;胸主动脉和肾动脉血管内皮细胞SA-β-gal活性明显增加,表现为较强的β-gal染色。结论：高脂饮食引起大鼠胸主动脉和肾动脉粥样硬化改变,动脉血管内皮细胞衰老增加。     

Genome reorganization during cellular senescence

It was previously suggested that aging of dividing cells depends on the reorganization of the cell genome during the division cycle and is determined by chance, intrinsic properties of the genome and environmental factors. A considerable amount of evidence has accumulated supporting the hypothesis. This is reviewed in terms of the reorganization taking place at the different orders of DNA structure.     

Altered endocytosis in cellular senescence

Cellular senescence occurs in response to diverse stresses (e.g., telomere shortening, DNA damage, oxidative stress, oncogene activation). A growing body of evidence indicates that alterations in multiple components of endocytic pathways contribute to cellular senescence. Clathrin-mediated endocytosis (CME) and caveolae-mediated endocytosis (CavME) represent major types of endocytosis that are implicated in senescence. More recent research has also identified a chromatin modifier and tumor suppressor that contributes to the induction of senescence via altered endocytosis. Here, molecular regulators of aberrant endocytosis-induced senescence are reviewed and discussed in the context of their capacity to serve as senescence-inducing stressors or modifiers.     

Cigarette smoke induces cellular senescence

Chronic obstructive pulmonary disease (COPD) is the fourth leading cause of death in the United States, and cigarette smoking is the major risk factor for COPD. Fibroblasts play an important role in repair and lung homeostasis. Recent studies have demonstrated a reduced growth rate for lung fibroblasts in patients with COPD. In this study we examined the effect of cigarette smoke extract (CSE) on fibroblast proliferative capacity. We found that cigarette smoke stopped proliferation of lung fibroblasts and upregulated two pathways linked to cell senescence (a biological process associated with cell longevity and an inability to replicate), p53 and p16-retinoblastoma protein pathways. We compared a single exposure of CSE to multiple exposures over an extended time course. A single exposure to CSE led to cell growth inhibition at multiple phases of the cell cycle without killing the cells. The decrease in proliferation was accompanied by increased ATM, p53, and p21 activity. However, several important senescent markers were not present in the cells at an earlier time point. When we examined multiple exposures to CSE, we found that the cells had profound growth arrest, a flat and enlarged morphology, upregulated p16, and senescence-associated beta-galactosidase activity, which is consistent with a classic senescent phenotype. These observations suggest that while a single exposure to cigarette smoke inhibits normal fibroblast proliferation (required for lung repair), multiple exposures to cigarette smoke move cells into an irreversible state of senescence. This inability to repair lung injury may be an essential feature of emphysema.     

Reduced Geminin levels promote cellular senescence

Cellular senescence is a permanent out-of-cycle state regulated by molecular circuits acting during the G1 phase of the cell cycle. Cdt1 is a central regulator of DNA replication licensing acting during the G1 phase and it is negatively controlled by Geminin. Here, we characterize the cell cycle expression pattern of Cdt1 and Geminin during successive passages of primary fibroblasts and compare it to tumour-derived cell lines. Cdt1 and Geminin are strictly expressed in distinct subpopulations of young fibroblasts, similarly to cancer cells, with Geminin accumulating shortly after the onset of S phase. Cdt1 and Geminin are down-regulated when primary human and mouse fibroblasts undergo replicative or stress-induced senescence. RNAi-mediated Geminin knock-down in human cells enhances the appearance of phenotypic and molecular features of senescence. Mouse embryonic fibroblasts heterozygous for Geminin exhibit accelerated senescence compared to control fibroblasts. In contrast, ectopic expression of Geminin in mouse embryonic fibroblasts delays the appearance of the senescent phenotype. Taken together, our data suggest that changes in Geminin expression levels affect the establishment of senescence pathways.     

Negative growth effectors and cellular senescence

Current studies suggest a genetic program governs the lifespan of each organism. Using cellular senscence as a model system, components of this program for aging sought. Human diploid fibroblast, upon reaching senescence, express active inhibitors of DNA synthesis. It is believed that such inhibitors could be members of a new family of negative growth effectors involved in the pathway to senescence. Factors capable of inhibiting DNA synthesis in a similar manner have also been identified from human quiescent fibroblasts and liver cells as well as from quiescent rodent liver cells. The relationship of these inhibitors to previously identified negative growth effectors and aging are discussed.     

Histone gene stability during cellular senescence

The extent to which human histone gene organization is conserved during the in vitro lifespan of human diploid fibroblast-like cells was determined by comparing the restriction patterns of a human H4 and an H3 histone gene from cells of various in vitro ages. No age related change in the organization of these two genes was detected.     

Facilitation of dendritic mRNA transport by CPEB

In neurons, the proteins derived from mRNAs localized in dendrites have been implicated in synaptic plasticity. The cytoplasmic polyadenylation element (CPE), a cis element in the 3'-UTRs of specific dendritic mRNAs, promotes cytoplasmic polyadenylation-induced translation in response to synaptic stimulation. Here, we demonstrate that the CPE and its binding protein CPEB facilitate mRNA transport to dendrites. In rat hippocampal neurons infected with recombinant viruses, the CPE is sufficient to direct a reporter RNA into dendrites. CPEB-GFP protein forms RNA-containing particles that are transported into dendrites in a microtubule-dependent fashion at an average velocity of 4-8 microm/min. Such particles also contain maskin, a CPEB-associated factor that mediates cap-dependent translational repression of CPE-containing mRNA, and the molecular motors dynein and kinesin. Overexpression of CPEB in neurons promotes the transport of CPE-containing endogenous MAP2 mRNA to dendrites, whereas overexpression of a mutant CPEB that is defective for interaction with molecular motors inhibits this transport. In neurons derived from CPEB knockout mice, the dendritic transport of a CPE-containing reporter RNA is reduced. These results suggest a mechanism whereby CPE-containing mRNAs can be transported to dendrites in a translationally dormant form, but activated at synapses in response to NMDA receptor stimulation.     

High-affinity Na-dependent dicarboxylate cotransporter promotes cellular senescence by inhibiting SIRT1

High-affinity Na⁺-dependent dicarboxylate cotransporter (NaDC3) can transport Krebs cycle intermediates into cells. Our previous study has shown that NaDC3 promotes cellular senescence, but its mechanism is not clear. It is known that when the concentration of intermediates in Krebs cycle is increased, NAD⁺/NADH ratio will be decreased. NAD⁺-dependent histone deacetylase sirtuin1 (SIRT1) prolongs mammalian cellular lifespan. Therefore, we propose that NaDC3 accelerates cellular aging by inhibiting SIRT1. After NaDC3 was overexpressed in two human embryo lung fibroblastic cell lines, WI38 and MRC-5, we found that the cells displayed aging-related phenotypes in advance. Meanwhile, the level of SIRT1 activity was down-regulated. In WI38/hNaDC3 cells treated with the activators of SIRT1, aging-related phenotypes induced by NaDC3 were obviously improved. The NAD⁺/NADH ratio in WI38/hNaDC3 cells was also decreased. Further study found that enhanced intracellular NAD⁺ level could attenuate the aging phenotypes induced by NaDC3. Thus, NaDC3 promotes cellular senescence probably by inhibiting NAD⁺-dependent SIRT1.     

Connecting chaperone-mediated autophagy dysfunction to cellular senescence

Chaperone-mediated autophagy (CMA) is one of the main pathways of the lysosome-autophagy proteolytic system. It regulates different cellular process through the selective degradation of cytosolic proteins. In ageing, the function of CMA is impaired causing an inefficient stress response and the accumulation of damaged, oxidized or misfolded proteins, which is associated with numerous age-related diseases. Deficient protein degradation alters cellular proteostasis and activates signaling pathways that culminate in the induction of cellular senescence, whose accumulation is a typical feature of ageing. However, the relationship between CMA activity and cellular senescence has been poorly studied. Here, we review and integrate evidence showing that CMA dysfunction correlates with the acquisition of many hallmarks of cellular senescence and propose that loss of CMA function during aging promotes cellular senescence.     

MicroRNAs as a novel cellular senescence regulator

Cellular senescence is a program activated in normal cells in response to various types of stresses and is manifested by permanent arrest of cell cycle. Cellular senescence is closely related to tumor suppression, and may contribute to the ageing of organisms. The complex senescence cell phenotype has many different mechanisms. Recent studies have provided important insights regarding the role played by miRNAs during cellular senescence as a novel molecular mechanism. In this article, we will review the latest advances in the identification and validation of senescence-regulatory miRNAs and the possible mechanisms.     

Impact of cellular senescence signature on ageing research

Cellular senescence as the state of permanent inhibition of cell proliferation is a tumour-suppressive mechanism. However, due to the associated secretory phenotype senescence can also contribute to cancer and possibly other age-related diseases, such as obesity, diabetes, atherosclerosis and Alzheimer's disease. There are two major mechanisms of cellular senescence; replicative senescence depends on telomere erosion or dysfunction whilst stress-induced premature senescence (SIPS) is telomere-independent and also includes oncogene-induced senescence (OIS). The senescence phenotype is characterised by altered cellular morphology, increased activity for senescence-associated-β-galactosidase (SA-β-GAL), increased formation of senescence-associated heterochromatin foci (SAHF) and promyelocytic leukemia protein nuclear bodies (PML NBs), permanent DNA damage, chromosomal instability and an inflammatory secretome. Some of these markers have been identified in cells from age-related pathologies. However, to improve our understanding of the contribution of cellular senescence to organismal ageing and age-related disease, it is imperative to define an unequivocal signature of cellular senescence that is functionally connected with normal and pathological ageing. Herein, we describe the processes leading to senescence, and the current biomarkers of cellular senescence, with particular emphasis on the causal role of DNA damage responses involved in the process. We highlight the gaps in our knowledge both of the processes leading to senescence, and the signature of cellular senescence both in vitro and in vivo. A well-defined set of senescence biomarkers for ageing and age-related disease would have a strong impact on the diagnosis, staging and predicted outcomes of age-related disease, providing the basis for a pharmacological intervention to postpone ageing and age-related disease.     

Cell fusion induced by ERVWE1 or measles virus causes cellular senescence

Cellular senescence limits proliferation of potentially detrimental cells, preventing tumorigenesis and restricting tissue damage. However, the function of senescence in nonpathological conditions is unknown. We found that the human placental syncytiotrophoblast exhibited the phenotype and expressed molecular markers of cellular senescence. During embryonic development, ERVWE1-mediated cell fusion results in formation of the syncytiotrophoblast, which serves as the maternal/fetal interface at the placenta. Expression of ERVWE1 caused cell fusion in normal and cancer cells, leading to formation of hyperploid syncytia exhibiting features of cellular senescence. Infection by the measles virus, which leads to cell fusion, also induced cellular senescence in normal and cancer cells. The fused cells activated the main molecular pathways of senescence, the p53- and p16–pRb-dependent pathways; the senescence-associated secretory phenotype; and immune surveillance-related proteins. Thus, fusion-induced senescence might be needed for proper syncytiotrophoblast function during embryonic development, and reuse of this senescence program later in life protects against pathological expression of endogenous fusogens and fusogenic viral infections.