TGF-beta1 Does Not Induce Senescence of Multipotent Mesenchymal Stromal Cells and Has Similar Effects in Early and Late Passages

Transforming growth factor-beta 1 (TGF-β1) stimulates a broad range of effects which are cell type dependent, and it has been suggested to induce cellular senescence. On the other hand, long-term culture of multipotent mesenchymal stromal cells (MSCs) has a major impact on their cellular physiology and therefore it is well conceivable that the molecular events triggered by TGF-β1 differ considerably in cells of early and late passages. In this study, we analyzed the effect of TGF-β1 on and during replicative senescence of MSCs. Stimulation with TGF-β1 enhanced proliferation, induced a network like growth pattern and impaired adipogenic and osteogenic differentiation. TGF-β1 did not induce premature senescence. However, due to increased proliferation rates the cells reached replicative senescence earlier than untreated controls. This was also evident, when we analyzed senescence-associated DNA-methylation changes. Gene expression profiles of MSCs differed considerably at relatively early (P 3 - 5) and later passages (P 10). Nonetheless, relative gene expression differences provoked by TGF-β1 at individual time points or in a time course dependent manner (stimulation for 0, 1, 4 and 12 h) were very similar in MSCs of early and late passage. These results support the notion that TGF-β1 has major impact on MSC function, but it does not induce senescence and has similar molecular effects during culture expansion.     

A potential role for NEDD1 and the centrosome in senescence of mouse embryonic fibroblasts

Mouse embryonic fibroblasts (MEFs) are commonly grown in cell culture and are known to enter senescence after a low number of passages as a result of oxidative stress. Oxidative stress has also been suggested to promote centrosome disruption; however, the contribution of this organelle to senescence is poorly understood. Therefore, this study aimed to assess the role of the centrosome in oxidative stress induced-senescence using MEFs as a model. We demonstrate here that coincident with the entry of late-passage MEFs into senescence, there was an increase in supernumerary centrosomes, most likely due to centrosome fragmentation. In addition, disrupting the centrosome in early-passage MEFs by depletion of neural precursor cell expressed developmentally downregulated gene 1 (NEDD1) also resulted in centrosomal fragmentation and subsequent premature entry into senescence. These data show that a loss of centrosomal integrity may contribute to the entry of MEFs into senescence in culture, and that centrosomal disruption can cause senescence.     

Increased abundance of cytoplasmic and nuclear caveolin 1 in human diploid fibroblasts in H(2)O(2)-induced premature senescence and interplay with p38alpha(MAPK)

Treatment of IMR-90 human diploid fibroblasts with a sublethal concentration of H(2)O(2) induces premature senescence. We investigated the protein abundance, subcellular localization and involvement of caveolin 1 in premature senescence. Caveolin 1 is a scaffolding protein able to concentrate and organize signaling molecules within the caveolae membrane domains. We report the first evidence of increased nuclear and cytoplasmic localization of caveolin 1 during establishment of H(2)O(2)-induced premature senescence. Moreover, we demonstrate that phosphorylation of caveolin 1 during treatment with H(2)O(2) is dependent on p38alpha mitogen-activated protein kinase.     

Inonotus obliquus protects against oxidative stress-induced apoptosis and premature senescence

In this study, we investigated the cytoprotective effects of Inonotus obliquus against oxidative stress-induced apoptosis and premature senescence. Pretreatment with I. obliquus scavenged intracellular ROS and prevented lipid peroxidation in hydrogen peroxide-treated human fibroblasts. As a result, I. obliquus exerted protective effects against hydrogen peroxide-induced apoptosis and premature senescence in human fibroblasts. In addition, I. obliquus suppressed UV-induced morphologic skin changes, such as skin thickening and wrinkle formation, in hairless mice in vivo and increased collagen synthesis through inhibition of MMP-1 and MMP-9 activities in hydrogen peroxide-treated human fibroblasts. Taken together, these results demonstrate that I. obliquus can prevent the aging process by attenuating oxidative stress in a model of stress-induced premature senescence.     

Comparison of global DNA methylation profiles in replicative versus premature senescence

DNA methylation is considered to play an essential role in cellular senescence. To uncover the mechanism underlying cellular senescence, we established the model of premature senescence induced by hydrogen peroxide (H(2)O(2)) in human embryonic lung fibroblasts and investigated the changes of genome methylation, DNA methyltransferases (DNMTs) and DNA-binding domain proteins (MBDs) in comparison with those observed during normal replicative senescence. We found that premature senescence triggered by H(2)O(2) exhibited distinct morphological characteristics and proliferative capacity which were similar to those of replicative senescence. The genome methylation level decreased gradually during the premature as well as replicative senescence, which was associated with the reduction in the expression of DNMT1, reflecting global hypomethylation as a distinct feature of senescent cells. The levels of DNMT3b and methyl-CpG binding protein 2 (MeCP2) increased in both mid-aged and replicative senescent cells, while DNMT3a and MBD2 were upregulated in the mid-aged cells. Only DNMT3b was elevated in the cells in the premature senescence persistence status. Additionally, the expression for DNMTs, MBD2 and MeCP2 was increased rapidly upon H(2)O(2) treatment. These results indicate that H(2)O(2)-induced premature senescence share some features of replicative senescence, such as basic biological characteristics and global hypomethylation while there are slight differences in the profile of methylation-associated enzyme expression. Oxidative damage may hence be a causative factor in epigenetic alteration partly responsible for cellular senescence.     

IL-6 Secreted from Senescent Mesenchymal Stem Cells Promotes Proliferation and Migration of Breast Cancer Cells

Human mesenchymal stem cells (hMSCs) are currently investigated for a variety of therapeutic applications. However, MSCs isolated from primary tissue cannot meet clinical grade needs and should be expanded in vitro for several passages. Although hMSCs show low possibility for undergoing oncogenic transformation, they do, similar to other somatic cells, undergo cellular senescence and their therapeutic potential is diminished when cultured in vitro. However, the role of senescent MSCs in tumor progression remains largely elusive. In the current study, by establishing senescent human umbilical cord mesenchymal stem cells (s-UCMSCs) through the replicative senescence model and genotoxic stress induced premature senescence model, we show that s-UCMSCs significantly stimulate proliferation and migration of breast cancer cells in vitro and tumor progression in a co-transplant xenograft mouse model compared with ‘young’ counterparts (defined as MSCs at passage 5, in contrast to senescent MSCs at passage 45). In addition, we identified IL-6, a known pleiotropic cytokine, as a principal mediator for the tumor-promoting activity of s-UCMSCs by induction of STAT3 phosphorylation. Depletion of IL-6 from s-UCMSCs conditioned medium partially abrogated the stimulatory effect of s-UCMSCs on the proliferation and migration of breast tumor cells.     

H2O2诱导的2BS早老细胞中自噬体增多

自噬在细胞复制性衰老中起着重要的作用.然而,早老细胞中的自噬现象基本无相关的报道.本文通过外源性过氧化氢(H₂O₂)的诱导,构建人胚肺二倍体成纤维细胞（2BS细胞）早老模型.首先,通过SA-β-gal染色,验证细胞早老;从形态学和特异标志分子及雷帕霉素作用的靶位点(mTOR)信号通路不同角度检测自噬的变化,其中形态学检测包括丹（磺）酰戊二胺(MDC)自噬分子定量法及电镜自噬超微结构的观察;特异标志分子LC3的检测包括GFP-LC3自噬定位法和免疫印迹法检测LC3;及检测mTOR信号通路下游激酶p70S6蛋白的表达变化.结果表明,过氧化氢诱导的早老细胞中自噬体相对年轻细胞明显增多,且具有保护早老细胞的作用.     

BMI-1026 treatment can induce SAHF formation by activation of Erk1/2

BMI-1026 is a synthetic aminopyrimidine compound that targets cyclin dependent kinases (cdks) and was initially designed as a potential anticancer drug. Even though it has been well documented that BMI-1026 is a potent cdk inhibitor, little is known about the cellular effects of this compound. In this study, we examined the effects of BMI-1026 treatment on inducing premature senescence and then evaluated the biochemical features of BMI-1026-induced premature senescence. From these experiments we determined that BMI-1026 treatment produced several biochemical features of premature senescence and also stimulated expression of mitogen activated protein kinase (MAPK) family proteins. BMI-1026 treatment caused nuclear translocation of activated Erk1/2 and the formation of senescence associated heterochromatin foci in 5 days. The heterochromatin foci formation was perturbed by inhibition of Erk1/2 activation.     

Human lipodystrophies linked to mutations in A-type lamins and to HIV protease inhibitor therapy are both associated with prelamin A accumulation, oxidative stress and premature cellular senescence

Lipodystrophic syndromes associated with mutations in LMNA, encoding A-type lamins, and with HIV antiretroviral treatments share several clinical characteristics. Nuclear alterations and prelamin A accumulation have been reported in fibroblasts from patients with LMNA mutations and adipocytes exposed to protease inhibitors (PI). As genetically altered lamin A maturation also results in premature ageing syndromes with lipodystrophy, we studied prelamin A expression and senescence markers in cultured human fibroblasts bearing six different LMNA mutations or treated with PIs. As compared to control cells, fibroblasts with LMNA mutations or treated with PIs had nuclear shape abnormalities and reduced proliferative activity that worsened with increasing cellular passages. They exhibited prelamin A accumulation, increased oxidative stress, decreased expression of mitochondrial respiratory chain proteins and premature cellular senescence. Inhibition of prelamin A farnesylation prevented cellular senescence and oxidative stress. Adipose tissue samples from patients with LMNA mutations or treated with PIs also showed retention of prelamin A, overexpression of the cell cycle checkpoint inhibitor p16 and altered mitochondrial markers. Thus, both LMNA mutations and PI treatment result in accumulation of farnesylated prelamin A and oxidative stress that trigger premature cellular senescence. These alterations could participate in the pathophysiology of lipodystrophic syndromes and lead to premature ageing complications.     

Tiller number is altered in the ascorbic acid-deficient rice suppressed for l-galactono-1,4-lactone dehydrogenase

The tiller of rice (Oryza sativa L.), which determines the panicle number per plant, is an important agronomic trait for grain production. Ascorbic acid (Asc) is a major plant antioxidant that serves many functions in plants. l-Galactono-1,4-lactone dehydrogenase (GLDH, EC 1.3.2.3) is an enzyme that catalyzes the last step of Asc biosynthesis in plants. Here we show that the GLDH-suppressed transgenic rices, GI-1 and GI-2, which have constitutively low (between 30% and 50%) leaf Asc content compared with the wild-type plants, exhibit a significantly reduced tiller number. Moreover, lower growth rate and plant height were observed in the Asc-deficient plants relative to the trait values of the wild-type plants at different tillering stages. Further examination showed that the deficiency of Asc resulted in a higher lipid peroxidation, a loss of chlorophyll, a loss of carotenoids, and a lower rate of CO₂ assimilation. In addition, the level of abscisic acid was higher in GI-1 plants, while the level of jasmonic acid was higher in GI-1 and GI-2 plants at different tillering stages. The results we presented here indicated that Asc deficiency was likely responsible for the promotion of premature senescence, which was accompanied by a marked decrease in photosynthesis. These observations support the conclusion that the deficiency of Asc alters the tiller number in the GLDH-suppressed transgenics through promoting premature senescence and changing phytohormones related to senescence.     

UVB-induced premature senescence of human diploid skin fibroblasts

In this work, we show that repeated stresses with UVB (290-320 nm) induce stress-induced premature senescence (SIPS) of skin human diploid fibroblasts (HDFs). HDFs at early cumulative population doublings were exposed three or five times to increasing subcytotoxic doses of UVB with one stress per day. After 2 days of recovery, several biomarkers of replicative senescence were established. First, there was an increase in the proportion of cells positive for senescence-associated beta-galactosidase activity. Second, there was a loss of replicative potential as assessed by a very low level of [3H]-thymidine incorporation. Third, the steady-state level of the mRNA of three senescence-associated genes, i.e. fibronectin, osteonectin and SM22, was increased in HDFs at 72 h after three and five exposures to UVB. In conclusion, these results suggest that it is possible to induce SIPS in HDFs after repeated exposures to subcytotoxic doses of UVB. This model could be used to test whether HDFs in UVB-induced premature senescence are able to promote epithelial cell growth and tumorigenesis in skin, as shown recently with HDFs in H(2)O(2)-induced premature senescence.     

Identification of 30 protein species involved in replicative senescence and stress-induced premature senescence

Exposure of human proliferative cells to subcytotoxic stress triggers stress-induced premature senescence (SIPS) which is characterized by many biomarkers of replicative senescence. Proteomic comparison of replicative senescence and stress-induced premature senescence indicates that, at the level of protein expression, stress-induced premature senescence and replicative senescence are different phenotypes sharing however similarities. In this study, we identified 30 proteins showing changes of expression level specific or common to replicative senescence and/or stress-induced premature senescence. These changes affect different cell functions, including energy metabolism, defense systems, maintenance of the redox potential, cell morphology and transduction pathways.     

Cisplatin-induced premature senescence with concomitant reduction of gap junctions in human fibroblasts

To examine the role of gap junctions in cell senescence, the changes of gap junctions in cisplatin-induced premature senescence of primary cultured fibroblasts were studied and compared with the replicative senescent human fibroblasts.Dye transfer assay for gap junction function and immunofluorescent staining for connexin 43 protein distribution were done respectively. Furthermore, cytofluorimetry and DAPI fluorescence staining were performed for cell cycle and apoptosis analysis, p53 gene expression level was detected with indirect immunofluorescence. We found that cisplatin(10mM) treatment could block cell growth cycle at G1 and induced premature senescence. The premature senescence changes included high frequency of apoptosis, elevation of p53 expression, loss of membranous gap junctions and reduction of dye-transfer capacity. These changes were comparable to the changes of replicative senescence of human fibroblasts. It was also concluded that cisplatin could induce premature senescence concomitant with inhibition of gap junctions in the fibroblasts. Loss of functional gap junctions from the cell membrane may account for the reduced intercellular communication in the premature senescent fibroblasts. The cell system we used may provide a model useful for the study of the gap junction thus promoting agents against premature senescence.     

Inhibition of the cGAS‐STING pathway ameliorates the premature senescence hallmarks of Ataxia‐Telangiectasia brain organoids

Ataxia‐telangiectasia (A‐T) is a genetic disorder caused by the lack of functional ATM kinase. A‐T is characterized by chronic inflammation, neurodegeneration and premature ageing features that are associated with increased genome instability, nuclear shape alterations, micronuclei accumulation, neuronal defects and premature entry into cellular senescence. The causal relationship between the detrimental inflammatory signature and the neurological deficiencies of A‐T remains elusive. Here, we utilize human pluripotent stem cell‐derived cortical brain organoids to study A‐T neuropathology. Mechanistically, we show that the cGAS‐STING pathway is required for the recognition of micronuclei and induction of a senescence‐associated secretory phenotype (SASP) in A‐T olfactory neurosphere‐derived cells and brain organoids. We further demonstrate that cGAS and STING inhibition effectively suppresses self‐DNA‐triggered SASP expression in A‐T brain organoids, inhibits astrocyte senescence and neurodegeneration, and ameliorates A‐T brain organoid neuropathology. Our study thus reveals that increased cGAS and STING activity is an important contributor to chronic inflammation and premature senescence in the central nervous system of A‐T and constitutes a novel therapeutic target for treating neuropathology in A‐T patients.     

Death-associated protein 3 regulates cellular senescence through oxidative stress response

Death-associated protein 3 (DAP3) has been originally identified as a positive mediator of apoptosis. It has been revealed recently that the predominant localization of DAP3 to mitochondria implies its functional involvement in mitochondrial metabolism in addition to apoptosis. However, little is known about the molecular basis of these physiological functions of DAP3. Here, we demonstrate that DAP3 is reduced in both replicative and premature senescence induced by oxidative stress, and the DAP3 reduction induced by oxidative stress is observed mostly in a mitochondrial fraction. Using DAP3-specific short hairpin RNA (shRNA) in a clonogenic survival assay, we reveal that reduction of DAP3 induces resistance to oxidative stress and decreases intracellular reactive oxygen species (ROS) production. Furthermore, this strategy allows us to show that loss of DAP3 is involved in the avoidance of replicative senescence in mouse embryonic fibroblasts (MEFs). Thus, our study offers an insight into the potential regulatory function of mitochondrial DAP3 involved in cellular senescence.     

The JAK1/2 inhibitor ruxolitinib delays premature aging phenotypes

Hutchinson–Gilford progeria syndrome (HGPS) is caused by an LMNA mutation that results in the production of the abnormal progerin protein. Children with HGPS display phenotypes of premature aging and have an average lifespan of 13 years. We found earlier that the targeting of the transmembrane protein PLA2R1 overcomes senescence and improves phenotypes in a mouse model of progeria. PLA2R1 is regulating the JAK/STAT signaling, but we do not yet know whether targeting this pathway directly would influence cellular and in vivo progeria phenotypes. Here, we show that JAK1/2 inhibition with ruxolitinib rescues progerin‐induced cell cycle arrest, cellular senescence, and misshapen nuclei in human normal fibroblasts expressing progerin. Moreover, ruxolitinib administration reduces several premature aging phenotypes: bone fractures, bone mineral content, grip strength, and a trend to increase survival in a mouse model of progeria. Thus, we propose that ruxolitinib, an FDA‐approved drug, should be further evaluated as a drug candidate in HGPS therapy.     

Senescence induced by RECQL4 dysfunction contributes to Rothmund–Thomson syndrome features in mice

Cellular senescence refers to irreversible growth arrest of primary eukaryotic cells, a process thought to contribute to aging-related degeneration and disease. Deficiency of RecQ helicase RECQL4 leads to Rothmund–Thomson syndrome (RTS), and we have investigated whether senescence is involved using cellular approaches and a mouse model. We first systematically investigated whether depletion of RECQL4 and the other four human RecQ helicases, BLM, WRN, RECQL1 and RECQL5, impacts the proliferative potential of human primary fibroblasts. BLM-, WRN- and RECQL4-depleted cells display increased staining of senescence-associated β-galactosidase (SA-β-gal), higher expression of p16^INK4a or/and p21^WAF1 and accumulated persistent DNA damage foci. These features were less frequent in RECQL1- and RECQL5-depleted cells. We have mapped the region in RECQL4 that prevents cellular senescence to its N-terminal region and helicase domain. We further investigated senescence features in an RTS mouse model, Recql4-deficient mice (Recql4^HD). Tail fibroblasts from Recql4^HD showed increased SA-β-gal staining and increased DNA damage foci. We also identified sparser tail hair and fewer blood cells in Recql4^HD mice accompanied with increased senescence in tail hair follicles and in bone marrow cells. In conclusion, dysfunction of RECQL4 increases DNA damage and triggers premature senescence in both human and mouse cells, which may contribute to symptoms in RTS patients.