Klotho RNAi induces premature senescence of human cells via a p53/p21 dependent pathway

Klotho has recently emerged as a regulator of aging. To investigate the role of Klotho in the regulation of cellular senescence, we generated stable MRC-5 human primary fibroblast cells knockdown for Klotho expression by RNAi. Downregulation of Klotho dramatically induces premature senescence with a concomitant upregulation of p21. The upregulation of p21 is associated with cell cycle arrest at G1/S boundary. Knockdown of p53 in the Klotho attenuated MRC-5 cells restores normal growth and replicative potential. These results demonstrate that Klotho normally regulates cellular senescence by repressing the p53/p21 pathway. Our findings implicate Klotho as a regulator of aging in primary human fibroblasts.     

ROS leads to MnSOD upregulation through ERK2 translocation and p53 activation in selenite-induced apoptosis of NB4 cells

Following our previous finding that sodium selenite induces apoptosis in human leukemia NB4 cells, we now show that the expression of the critical antioxidant enzyme manganese superoxide dismutase (MnSOD) is remarkably elevated during this process. We further reveal that reactive oxygen species (ROS), especially superoxide radicals, play a crucial role in selenite-induced MnSOD upregulation, with extracellular regulated kinase (ERK) and p53 closely implicated. Specifically, ERK2 translocates into the nucleus driven by ROS, where it directly phosphorylates p53, leading to dissociation of p53 from its inhibitory protein mouse double minute 2 (MDM2). Active p53 directly mediates the expression of MnSOD, serving as the link between ERK2 translocation and MnSOD upregulation.     

Autophagy impairment induces premature senescence in primary human fibroblasts

Background

Recent studies have demonstrated that activation of autophagy increases the lifespan of organisms from yeast to flies. In contrast to the lifespan extension effect in lower organisms, it has been reported that overexpression of unc-51-like kinase 3 (ULK3), the mammalian homolog of autophagy-specific gene 1 (ATG1), induces premature senescence in human fibroblasts. Therefore, we assessed whether the activation of autophagy would genuinely induce premature senescence in human cells.

Methodology/Principal Findings

Depletion of ATG7, ATG12, or lysosomal-associated membrane protein 2 (Lamp2) by transfecting siRNA or infecting cells with a virus containing gene-specific shRNA resulted in a senescence-like state in two strains of primary human fibroblasts. Prematurely senescent cells induced by autophagy impairment exhibited the senescent phenotypes, similar to the replicatively senescent cells, such as increased senescence associated β-galactosidase (SA-β-gal) activity, reactive oxygen species (ROS) generation, and accumulation of lipofuscin. In addition, expression levels of ribosomal protein S6 kinase1 (S6K1), p-S6K1, p-S6, and eukaryotic translation initiation factor 4E (eIF4E) binding protein 1 (4E-BP1) in the mammalian target of rapamycin (mTOR) pathway and beclin-1, ATG7, ATG12-ATG5 conjugate, and the sequestosome 1 (SQSTM1/p62) monomer in the autophagy pathway were decreased in both the replicatively and the autophagy impairment-induced prematurely senescent cells. Furthermore, it was found that ROS scavenging by N-acetylcysteine (NAC) and inhibition of p53 activation by pifithrin-α or knockdown of p53 using siRNA, respectively, delayed autophagy impairment-induced premature senescence and restored the expression levels of components in the mTOR and autophagy pathways.

Conclusion

Taken together, we concluded that autophagy impairment induces premature senescence through a ROS- and p53-dependent manner in primary human fibroblasts.     

The canonical NF-κB pathway differentially protects normal and human tumor cells from ROS-induced DNA damage

DNA damage responses (DDR) invoke senescence or apoptosis depending on stimulus intensity and the degree of activation of the p53-p21(Cip1/Waf1) axis; but the functional impact of NF-κB signaling on these different outcomes in normal vs. human cancer cells remains poorly understood. We investigated the NF-κB-dependent effects and mechanism underlying reactive oxygen species (ROS)-mediated DDR outcomes of normal human lung fibroblasts (HDFs) and A549 human lung cancer epithelial cells. To activate DDR, ROS accumulation was induced by different doses of H(2)O(2). The effect of ROS induction caused a G2 or G2-M phase cell cycle arrest of both human cell types. However, ROS-mediated DDR eventually culminated in different end points with HDFs undergoing premature senescence and A549 cancer cells succumbing to apoptosis. NF-κB p65/RelA nuclear translocation and Ser536 phosphorylation were induced in response to H(2)O(2)-mediated ROS accumulation. Importantly, blocking the activities of canonical NF-κB subunits with an IκBα super-repressor or suppressing canonical NF-κB signaling by IKKβ knock-down accelerated HDF premature senescence by up-regulating the p53-p21(Cip1/Waf1) axis; but inhibiting the canonical NF-κB pathway exacerbated H(2)O(2)-induced A549 cell apoptosis. HDF premature aging occurred in conjunction with γ-H2AX chromatin deposition, senescence-associated heterochromatic foci and beta-galactosidase staining. p53 knock-down abrogated H(2)O(2)-induced premature senescence of vector control- and IκBαSR-expressing HDFs functionally linking canonical NF-κB-dependent control of p53 levels to ROS-induced HDF senescence. We conclude that IKKβ-driven canonical NF-κB signaling has different functional roles for the outcome of ROS responses in the contexts of normal vs. human tumor cells by respectively protecting them against DDR-dependent premature senescence and apoptosis.     

Absence of superoxide dismutase activity causes nuclear DNA fragmentation during the aging process

Superoxide dismutases (SOD) serve as an important antioxidant defense mechanism in aerobic organisms, and deletion of these genes shortens the replicative life span in the budding yeast Saccharomyces cerevisiae. Even though involvement of superoxide dismutase enzymes in ROS scavenging and the aging process has been studied extensively in different organisms, analyses of DNA damages has not been performed for replicatively old superoxide dismutase deficient cells. In this study, we investigated the roles of SOD1, SOD2 and CCS1 genes in preserving genomic integrity in replicatively old yeast cells using the single cell comet assay. We observed that extend of DNA damage was not significantly different among the young cells of wild type, sod1Δ and sod2Δ strains. However, ccs1Δ mutants showed a 60% higher amount of DNA damage in the young stage compared to that of the wild type cells. The aging process increased the DNA damage rates 3-fold in the wild type and more than 5-fold in sod1Δ, sod2Δ, and ccs1Δ mutant cells. Furthermore, ROS levels of these strains showed a similar pattern to their DNA damage contents. Thus, our results confirm that cells accumulate DNA damages during the aging process and reveal that superoxide dismutase enzymes play a substantial role in preserving the genomic integrity in this process.     

Influence of induced reactive oxygen species in p53-mediated cell fate decisions

The p53 tumor suppressor gene can induce either apoptosis or a permanent growth arrest (also termed senescence) phenotype in response to cellular stresses. We show that the increase in intracellular reactive oxygen species (ROS) associated with the magnitude of p53 protein expression correlated with the induction of either senescence or apoptosis in both normal and cancer cells. ROS inhibitors ameliorated both p53-dependent cell fates, implicating ROS accumulation as an effector in each case. The absence of Bax or PUMA strongly inhibited both p53-induced apoptosis and ROS increase, indicating an important role these p53 targets affecting mitochondrial function genes in p53-mediated ROS accumulation. Moreover, physiological p53 levels in combination with an exogenous ROS source were able to convert a p53 senescence response into apoptosis. All of these findings establish a critical role of ROS accumulation and mitochondrial function in p53-dependent cell fates and show that other ROS inducers can collaborate with p53 to influence these fate decisions. Thus, our studies imply that therapeutic agents that generate ROS are more likely to be toxic for normal cells than p53-negative tumor cells and provide a rationale for identifying therapeutic agents that do not complement p53 in ROS generation to ameliorate the cytotoxic side effects in normal cells.     

p21Waf1/Cip1 plays a critical role in modulating senescence through changes of DNA methylation

It has been reported that genomic DNA methylation decreases gradually during cell culture and an organism's aging. However, less is known about the methylation changes of age-related specific genes in aging. p21(Waf1/Cip1) and p16(INK4a) are cyclin-dependent kinase (Cdk) inhibitors that are critical for the replicative senescence of normal cells. In this study, we show that p21(Waf1/Cip1) and p16(INK4a) have different methylation patterns during the aging process of normal human 2BS and WI-38 fibroblasts. p21(Waf1/Cip1) promoter is gradually methylated up into middle-aged fibroblasts but not with senescent fibroblasts, whereas p16(INK4a) is always unmethylated in the aging process. Correspondently, the protein levels of DNA methyltransferase 1 (DNMT1) and DNMT3a increase from young to middle-aged fibroblasts but decrease in the senescent fibroblasts, while DNMT3b decreases stably from young to senescent fibroblasts. p21(Waf1/Cip1) promoter methylation directly represses its expression and blocks the radiation-induced DNA damage-signaling pathway by p53 in middle-aged fibroblasts. More importantly, demethylation by 5-aza-CdR or DNMT1 RNA interference (RNAi) resulted in an increased p21(Waf1/Cip1) level and premature senescence of middle-aged fibroblasts demonstrated by cell growth arrest and high beta-Galactosidase expression. Our results suggest that p21(Waf1/Cip1) but not p16(INK4a) is involved in the DNA methylation mediated aging process. p21(Waf1/Cip1) promoter methylation may be a critical biological barrier to postpone the aging process.     

Nek6 overexpression antagonizes p53-induced senescence in human cancer cells

Nek6 is an NIMA-related kinase that plays a critical role in mitotic cell cycle progression. Recent studies have shown that Nek6 is upregulated in various human cancers, but the function of Nek6 in tumorigenesis is largely unknown. Here, we examined the role of Nek6 in cellular senescence. Our data revealed that Nek6 expression is decreased both in both the replicative senescence of human normal fibroblasts and premature senescence induced by p53 expression in EJ human bladder cancer cells and H1299 human lung cancer cells. Interestingly, the enforced expression of Nek6 in EJ and H1299 cells completely suppresses p53-induced senescence, whereas the expression of kinase-dead Nek6 did not affect p53-induced senescence. Mechanistic studies revealed that cell cycle arrest in the G1 and G2/M phases, as well as the reduction of cyclin B and cdc2 protein level upon p53 expression were significantly reduced by Nek6 overexpression. In addition, p53-induced increases in intracellular levels of ROS were also inhibited in cells overexpressing Nek6. These results suggest that the downregulation of Nek6 expression is required for the onset of p53-induced cellular senescence and imply a possible role of Nek6 in tumorigenesis.     

Cooperation between p21 and Akt is required for p53‐dependent cellular senescence

Cellular senescence has been implicated in normal aging, tissue homeostasis, and tumor suppression. Although p53 has been shown to be a central mediator of cellular senescence, the signaling pathway by which it induces senescence remains incompletely understood. In this study, we have shown that both Akt and p21 are required to induce cellular senescence in response to p53 expression. In a p53‐induced senescence model, we found that Akt activation was essential for inducing a cellular senescence phenotype. Surprisingly, Akt inhibition did not abolish p53‐induced cell cycle arrest, but it suppressed the increase in intracellular reactive oxygen species (ROS) levels. The results of the cell cycle and morphological analysis suggest that p53 induced quiescence, not senescence, following Akt inhibition. Conversely, the inhibition of p21 induction abolished cell cycle arrest but did not affect the p53‐induced increase in ROS levels. Additionally, p21 and Akt separately controlled cell cycle arrest and ROS levels, respectively, during H‐Ras‐induced senescence in human normal fibroblasts. The mechanistic analysis revealed that Akt increased ROS levels through NOX4 induction, and increased Akt‐dependent NF‐κB binding to the NOX4 promoter is responsible for NOX4 induction upon p53 expression. We further showed that Akt activation upon p53 expression is mediated by mammalian target of rapamycin complex 2. In addition, p53‐mediated IL6 and IL8 induction was abrogated by Akt inhibition, suggesting that Akt activation is also required for the senescence‐associated secretory phenotype. Collectively, these results suggest that p53 simultaneously controls multiple pathways to induce cellular senescence through p21 and Akt.     

Reactive oxygen species and mitochondrial sensitivity to oxidative stress determine induction of cancer cell death by p21

p21(Waf1/Cip1/Sdi1) is a cyclin-dependent kinase inhibitor that mediates cell cycle arrest. Prolonged p21 up-regulation induces a senescent phenotype in normal and cancer cells, accompanied by an increase in intracellular reactive oxygen species (ROS). However, it has been shown recently that p21 expression can also lead to cell death in certain models. The mechanisms involved in this process are not fully understood. Here, we describe an induction of apoptosis by p21 in sarcoma cell lines that is p53-independent and can be ameliorated with antioxidants. Similar levels of p21 and ROS caused senescence in the absence of significant death in other cancer cell lines, suggesting a cell-specific response. We also found that cells undergoing p21-dependent cell death had higher sensitivity to oxidants and a specific pattern of mitochondrial polarization changes. Consistent with this, apoptosis could be blocked with targeted expression of catalase in the mitochondria of these cells. We propose that the balance between cancer cell death and arrest after p21 up-regulation depends on the specific effects of p21-induced ROS on the mitochondria. This suggests that selective up-regulation of p21 in cancer cells could be a successful therapeutic intervention for sarcomas and tumors with lower resistance to mitochondrial oxidative damage, regardless of p53 status.     

NADPH oxidase is involved in protein kinase CKII down-regulation-mediated senescence through elevation of the level of reactive oxygen species in human colon cancer cells

We have shown that protein kinase CKII (CKII) inhibition induces senescence through the p53-dependent pathway in HCT116 cells. Here we examined the molecular mechanism through which CKII inhibition activates p53 in HCT116 cells. CKII inhibition by treatment with CKII inhibitor or CKIIα small-interfering RNA (siRNA) increased intracellular hydrogen peroxide and superoxide anion levels. These effects were significantly blocked by pretreatment of cells with the antioxidant N-acetylcysteine. Additionally, NADPH oxidase (NOX) inhibitor apocynin and p22^phox siRNA significantly reduced p53 expression and suppressed the appearance of senescence markers. CKII inhibition did not affect mitochondrial superoxide generation. These data demonstrate that CKII inhibition induces superoxide anion generation via NOX activation, and subsequent superoxide-dependent activation of p53 acts as a mediator of senescence in HCT116 cells after down-regulation of CKII.     

IGF-I enhances cellular senescence via the reactive oxygen species-p53 pathway

Cellular senescence is characterized by growth arrest, enlarged and flattened cell morphology, the expression of senescence-associated β-galactosidase (SA-β-gal), and by activation of tumor suppressor networks. Insulin-like growth factor-I (IGF-I) plays a critical role in cellular growth, proliferation, tumorigenesis, and regulation of aging. In the present study, we show that IGF-I enhances cellular senescence in mouse, rat, and human primary cells in the confluent state. IGF-I induced expression of a DNA damage marker, γH2AX, the increased levels of p53 and p21 proteins, and activated SA-β-gal. In the confluent state, an altered downstream signaling of IGF-I receptor was observed. Treatment with a reactive oxygen species (ROS) scavenger, N-acetylcystein (NAC) significantly suppressed induction of these markers, indicating that ROS are involved in the induction of cellular senescence by IGF-I. In p53-null mouse embryonic fibroblasts, the IGF-I-induced augmentation of SA-β-gal and p21 was inhibited, demonstrating that p53 is required for cellular senescence induced by IGF-I. Thus, these data reveal a novel pathway whereby IGF-I enhances cellular senescence in the ROS and p53-dependent manner and may explain the underlying mechanisms of IGF-I involvement in tumorigenesis and in regulation of aging.     

Suppression of thioredoxin-1 induces premature senescence in normal human fibroblasts

Thioredoxin (TRX) is a ubiquitous multifunctional thiol protein that is critically involved in maintaining cellular redox homeostasis. Levels of thioredoxin-1 (TRX1), the major isoform of TRX, have been shown to correlate with organismal lifespan and age-associated tissue deterioration. Accordingly, we investigated the direct functional effects of suppressing TRX1 levels on cellular senescence, a phenomenon intimately linked with tissue degeneration and aging. Here we find that suppression of TRX1 expression via shRNA rapidly induces premature senescence in young human skin fibroblasts through upregulation of the p53/p21^Cip1/Waf1 and p16^INK4a tumor suppressor pathways. Moreover, inhibition of these pathways by introduction of SV40 Large T Antigen prevents TRX1 suppression-induced premature senescence but not susceptibility to oxidative stressors. Thus our results suggest that TRX1 has a role in suppressing senescence in normal cells in addition to its function as a redox-protective protein.     

Partial proteasome inhibition in human fibroblasts triggers accelerated M1 senescence or M2 crisis depending on p53 and Rb status

Proteasome-dependent degradation has been extensively investigated and has been shown to play a vital role in the maintenance of cellular homeostasis. Proteasome activity and expression are reduced during aging and replicative senescence. Its activation has been shown to confer lifespan extension in human diploid fibroblasts (HDFs), whereas partial proteasome inhibition triggers an irreversible premature senescent state in young HDFs. As p53 and Rb tumor suppressors regulate both replicative and premature senescence (RS and PS, respectively), in this study we investigated their implication in proteasome inhibition-mediated PS. By taking advantage of a variety of HDFs with defective p53 or/and Rb pathways, we reveal that proteasome activity inhibition to levels normally found in senescent human cells results in immediate growth arrest and/or moderate increase of apoptotic death. These effects are independent of the cellular genetic context. However, in the long term, proteasome inhibition-mediated PS can only be initiated and maintained in the presence of functional p53. More specifically, we demonstrate that following partial proteasome inhibition, senescence is dominant in HDFs with functional p53 and Rb molecules, crisis/death is induced in cells with high p53 levels and defective Rb pathway, whereas stress recovery and restoration of normal cycling occurs in cells that lack functional p53. These data reveal the continuous interplay between the integrity of proteasome function, senescence and cell survival.     

Loss of proliferative capacity and induction of senescence in oxidatively stressed human fibroblasts

Cellular senescence can result from short, dysfunctional telomeres, oxidative stress, or oncogene expression, and may contribute to aging. To investigate the role of cellular senescence in aging it is necessary to define the time-dependent molecular events by which it is characterized. Here we investigated changes in levels of key proteins involved in cell cycle regulation, DNA replication, and stress resistance in senescing human fibroblasts following oxidative stress. An immediate response in stressed cells was dephosphorylation of retinoblastoma (Rb) and cessation of DNA synthesis. This was followed by sequential induction of p53, p21, and p16. Increase in hypophosphorylated Rb and induction of p53 and p21 by a single stress treatment was transient, whereas sustained induction or dephosphorylation were achieved by a second stress. Down-regulation of the critical DNA replication initiation factor Cdc6 occurred early after stress concurring with p53 induction, and was followed by a decrease in Mcm2 levels. A late event in the stress-induced molecular sequence was the induction of SOD1, catalase, and HSP27 coinciding with development of the fully senescent phenotype. Our data suggest that loss of proliferative capacity in oxidatively stressed cells is a multistep process regulated by time-dependent molecular events that may play differential roles in induction and maintenance of cellular senescence.     

Functional restoration of replicative senescent mesenchymal stem cells by the brown alga Undaria pinnatifida

The brown alga Undaria pinnatifida, which is called Mi-Yoek in Korea, has been traditionally consumed as a health food in East Asian countries. Recent studies have reported that U. pinnatifida has beneficial effects on arteriosclerosis, inflammation, fat metabolism, and tumors. In this study, we examined the anti-senescence effects of ethanol extracts of U. pinnatifida (UP-Ex) in human bone marrow mesenchymal stem cells (hBM-MSCs). UP-Ex protected hBM-MSCs against oxidative injury, as determined by MTT assays. This effect was confirmed by immunoblot analysis of the oxidation-sensitive protein p53 and the apoptotic protein cleaved caspase-3. Excessive intracellular reactive oxygen species (ROS) accumulation induced by oxidative stress was moderated in UP-Ex-treated hBM-MSCs (UP-Ex-MSCs). Similarly, expression of the ROS-scavenging enzymes superoxide dismutase 1 (SOD1), SOD2, and catalase was recovered in UP-Ex-MSCs. Excessive ROS induced by long-term cell expansion (passage 17) was significantly decreased along with restoration of the senescence proteins p53, p21, and p16 in UP-Ex-MSCs. UP-Ex treatment also improved the ability of these replicative, senescent hBM-MSCs (passage 17) to differentiate into osteocytes or adipocytes, suggesting that UP-Ex ameliorates the functional decline of senescent stem cells and may provide better therapeutic efficacy in stem cell therapy.

Abbreviations: hBM-MSCs: human bone marrow mesenchymal stem cells; DCF: 2′,7′-dichlorodihydrofluorescein; DCFH-DA: 2′,7′-dichlorofluorescein diacetate; MTT: 3-(4,5-dimethylthiazol-2-yl-)2,5-diphenyltetrazolium bromide; PBS: phosphate-buffered saline; PFA: paraformaldehyde; RIPA: radioimmunoprecipitation assay; ROS: reactive oxygen species; SOD1: superoxide dismutase 1; SOD2: superoxide dismutase 2.     

Iron chelation study in a normal human hepatocyte cell line suggests that tumor necrosis factor receptor-associated protein 1 (TRAP1) regulates production of reactive oxygen species

Iron is an essential component of many proteins, and has crucial roles in the proper functioning of proteins involved in cellular respiration, proliferation, and differentiation. It has been recently reported that the deferoxamine (DFO), an iron chelator, induces mitochondrial dysfunction, characterized by an attenuation of oxidative phosphorylation, as well as senescence-like cellular morphology. However, the effects of DFO on mitochondrial heat shock proteins (HSPs) remain poorly understood. In this study, we examined the effect of DFO on tumor necrosis factor receptor-associated protein 1 (TRAP1), a representative mitochondrial HSP, in a normal human hepatocyte cell line, Chang cells. DFO specifically decreased TRAP1 levels, increasing reactive oxygen species (ROS) and caveolin-1 (Cav-1), a marker protein of senescence. To examine whether these effects of DFO are reversed, we established TRAP1-overexpressing Chang cells. DFO treatment to TRAP1-overexpressing cells resulted in decreases in levels of ROS, Cav-1, glutathione peroxidase (GPX), and manganese superoxide dismutase (MnSOD) levels as well as senescence-associated beta-galactosidase (SA beta-gal) activity. These results suggest that TRAP1 might play a role in protecting mitochondria against damaging stimuli via decrease of ROS generation.     

Simvastatin rises reactive oxygen species levels and induces senescence in human melanoma cells by activation of p53/p21 pathway

Recent studies demonstrated that simvastatin has antitumor properties in several types of cancer cells, mainly by inducing apoptosis and inhibiting growth. The arrest of proliferation is a feature of cellular senescence; however, the occurrence of senescence in melanoma cells upon simvastatin treatment has not been investigated until now. Our results demonstrated that exposure of human metastatic melanoma cells (WM9) to simvastatin induces a senescent phenotype, characterized by G1 arrest, positive staining for senescence-associated β-galactosidase assay, and morphological changes. Also, the main pathways leading to cell senescence were examined in simvastatin-treated human melanoma cells, and the expression levels of phospho-p53 and p21 were upregulated by simvastatin, suggesting that cell cycle regulators and DNA damage pathways are involved in the onset of senescence. Since simvastatin can act as a pro-oxidant agent, and oxidative stress may be related to senescence, we measured the intracellular ROS levels in WM9 cells upon simvastatin treatment. Interestingly, we found an increased amount of intracellular ROS in these cells, which was accompanied by elevated expression of catalase and peroxiredoxin-1. Collectively, our results demonstrated that simvastatin can induce senescence in human melanoma cells by activation of p53/p21 pathway, and that oxidative stress may be related to this process.