Defective nuclear translocation of stress-activated signaling in senescent diploid human fibroblasts: a possible explanation for aging-associated apoptosis resistance

In order to study the nature of aging-dependent apoptosis resistance, we compared the activation pattern of mitogen-activated protein kinases (MAPK) in response to three different stress modalities: hydrogen peroxide (H₂O₂), staurosporine, and thapsigargin. We observed the agonist-specific activation pattern of MAP kinases in human diploid fibroblasts (HDFs). When young HDFs were treated with PD98059, a specific inhibitor of extracellular signal-regulated kinase (ERK), H₂O₂-induced apoptosis was blocked, whereas staurosporine-induced apoptosis was inhibited by treatment with SB203580, a specific inhibitor of p38. In addition, the levels of anti-apoptotic protein Bcl-2 (B-cell lymphoma protein-2) were restored by PD98059 or SB239063 in cells treated with H₂O₂ or staurosporine, respectively. We also found that inhibition of the nuclear import of p-Erk and p-p38 using wheat germ agglutinin induced apoptosis resistance in young HDF cells in response to H₂O₂ or staurosporine. These data indicate a potential role of the nuclear translocation of apoptotic signals in the induction of apoptosis. Moreover, the nuclear translocation of activated ERK1/2 and p38 in response to H₂O₂ or staurosporine was significantly compromised in senescent HDFs, compared with young cells. Taken together, we propose that the apoptosis resistance of senescent HDFs might be related to the defective nuclear translocation of stress-activated signals in an agonist-specific manner, which would imply the operation of an aging-dependent functional nucleo-cytoplasmic trafficking barrier.     

胸苷激酶基因在人二倍体成纤维细胞衰老过程中的表达调控

为研究人胸苷激酶 (humanthymidinekinase ,hTK)基因在复制衰老细胞及早衰细胞中表达下调的分子机制 ,构建了含hTK启动子的荧光素酶报告基因载体 .转染结果显示 ,复制衰老细胞与早衰细胞中hTK启动子的转录活性比年轻细胞中下降了近 3倍 ,表明转录水平的调控是hTK在衰老细胞中表达下降的主要调控机制 .定点突变的结果显示 ,转录因子Sp1、NF Y结合位点的突变可使hTK启动子活性降低近 5 0 % ,而E2F结合位点的突变可使其活性升高 2倍多 ,提示Sp1和NF Y是hTK基因的转录活化因子 ,而E2F为转录抑制因子 .电泳迁移率变更实验发现 ,与年轻细胞相比 ,Sp1、NF Y与hTK启动子的DNA结合活性在复制衰老细胞和早衰细胞中无明显改变 ,提示转录活化因子Sp1、NF Y并非hTK在衰老细胞中下调的主要因素 .染色质免疫共沉淀结果显示 ,在细胞内Rb结合在hTK启动子上 ,且同年轻细胞相比 ,复制衰老细胞及早衰细胞中的hTK启动子结合着更多的Rb ,这提示细胞衰老过程中Rb的去磷酸化可能与hTK基因在衰老过程中的下调有关 .     

Phenyl 2‐pyridyl ketoxime induces cellular senescence‐like alterations via nitric oxide production in human diploid fibroblasts

Phenyl‐2‐pyridyl ketoxime (PPKO) was found to be one of the small molecules enriched in the extracellular matrix of near‐senescent human diploid fibroblasts (HDFs). Treatment of young HDFs with PPKO reduced the viability of young HDFs in a dose‐ and time‐dependent manner and resulted in senescence‐associated β‐galactosidase (SA‐β‐gal) staining and G2/M cell cycle arrest. In addition, the levels of some senescence‐associated proteins, such as phosphorylated ERK1/2, caveolin‐1, p53, p16^ink4a, and p21^waf1, were elevated in PPKO‐treated cells. To monitor the effect of PPKO on cell stress responses, reactive oxygen species (ROS) production was examined by flow cytometry. After PPKO treatment, ROS levels transiently increased at 30 min but then returned to baseline at 60 min. The levels of some antioxidant enzymes, such as catalase, peroxiredoxin II and glutathione peroxidase I, were transiently induced by PPKO treatment. SOD II levels increased gradually, whereas the SOD I and III levels were biphasic during the experimental periods after PPKO treatment. Cellular senescence induced by PPKO was suppressed by chemical antioxidants, such as N‐acetylcysteine, 2,2,6,6‐tetramethylpiperidinyloxy, and L‐buthionine‐(S,R)‐sulfoximine. Furthermore, PPKO increased nitric oxide (NO) production via inducible NO synthase (iNOS) in HDFs. In the presence of NOS inhibitors, such as L‐NG‐nitroarginine methyl ester and L‐NG‐monomethylarginine, PPKO‐induced transient NO production and SA‐β‐gal staining were abrogated. Taken together, these results suggest that PPKO induces cellular senescence in association with transient ROS and NO production and the subsequent induction of senescence‐associated proteins .     

Induction of replicative senescence biomarkers by sublethal oxidative stresses in normal human fibroblast

We tested the long-term effects of sublethal oxidative stresses on replicative senescence. WI-38 human diploid fibroblasts (HDFs) at early cumulative population doublings (CPDs) were exposed to five stresses with 30 microM tert-butylhydroperoxide (t-BHP). After at least 2 d of recovery, the cells developed biomarkers of replicative senescence: loss of replicative potential, increase in senescence-associated beta-galactosidase activity, overexpression of p21(Waf-1/SDI-1/Cip1), and inability to hyperphosphorylate pRb. The level of mRNAs overexpressed in senescent WI-38 or IMR-90 HDFs increased after five stresses with 30 microM t-BHP or a single stress under 450 microM H(2)O(2). These corresponding genes include fibronectin, osteonectin, alpha1(I)-procollagen, apolipoprotein J, SM22, SS9, and GTP-alpha binding protein. The common 4977 bp mitochondrial DNA deletion was detected in WI-38 HDFs at late CPDs and at early CPDs after t-BHP stresses. In conclusion, sublethal oxidative stresses lead HDFs to a state close to replicative senescence.     

HO-1 nuclear accumulation and interaction with NPM1 protect against stress-induced endothelial senescence independent of its enzymatic activity

Heme oxygenase-1 (HO-1) has attracted accumulating attention for its antioxidant enzymatic activity. However, the exact regulatory role of its non-enzymatic activity in the cardiovascular system remains unaddressed. Here, we show that HO-1 was accumulated in the nuclei of stress-induced senescent endothelial cells, and conferred protection against endothelial senescence independent of its enzymatic activity. Overexpression of ΔHO-1, a truncated HO-1 without transmembrane segment (TMS), inhibited H₂O₂-induced endothelial senescence. Overexpression of ΔHO-1_H25A, the catalytically inactive form of ΔHO-1, also exhibited anti-senescent effect. In addition, infection of recombinant adenovirus encoding ΔHO-1 with three nuclear localization sequences (NLS), alleviated endothelial senescence induced by knockdown of endogenous HO-1 by CRISPR/Cas9. Moreover, repression of HO-1 nuclear translocation by silencing of signal peptide peptidase (SPP), which is responsible for enzymatic cleavage of the TMS of HO-1, exacerbated endothelial senescence. Mechanistically, nuclear HO-1 interacted with NPM1 N-terminal portion, prevented NPM1 translocation from nucleolus to nucleoplasm, thus disrupted NPM1/p53/MDM2 interactions and inhibited p53 activation by NPM1, finally resisted endothelial senescence. This study provides a novel understanding of HO-1 as a promising therapeutic strategy for vascular senescence-related cardiovascular diseases.Subject terms: Stress signalling, Vascular diseases     

Downregulation of the inflammatory network in senescent fibroblasts and aging tissues of the long‐lived and cancer‐resistant subterranean wild rodent,Spalax

The blind mole rat (Spalax) is a wild, long‐lived rodent that has evolved mechanisms to tolerate hypoxia and resist cancer. Previously, we demonstrated high DNA repair capacity and low DNA damage in Spalax fibroblasts following genotoxic stress compared with rats. Since the acquisition of senescence‐associated secretory phenotype (SASP) is a consequence of persistent DNA damage, we investigated whether cellular senescence in Spalax is accompanied by an inflammatory response. Spalax fibroblasts undergo replicative senescence (RS) and etoposide‐induced senescence (EIS), evidenced by an increased activity of senescence‐associated beta‐galactosidase (SA‐β‐Gal), growth arrest, and overexpression of p21, p16, and p53 mRNAs. Yet, unlike mouse and human fibroblasts, RS and EIS Spalax cells showed undetectable or decreased expression of the well‐known SASP factors: interleukin‐6 (IL6), IL8, IL1α, growth‐related oncogene alpha (GROα), SerpinB2, and intercellular adhesion molecule (ICAM‐1). Apparently, due to the efficient DNA repair in Spalax, senescent cells did not accumulate the DNA damage necessary for SASP activation. Conversely, Spalax can maintain DNA integrity during replicative or moderate genotoxic stress and limit pro‐inflammatory secretion. However, exposure to the conditioned medium of breast cancer cells MDA‐MB‐231 resulted in an increase in DNA damage, activation of the nuclear factor κB (NF‐κB) through nuclear translocation, and expression of inflammatory mediators in RS Spalax cells. Evaluation of SASP in aging Spalax brain and intestine confirmed downregulation of inflammatory‐related genes. These findings suggest a natural mechanism for alleviating the inflammatory response during cellular senescence and aging in Spalax, which can prevent age‐related chronic inflammation supporting healthy aging and longevity.     

Bradykinin Inhibits Oxidative Stress-Induced Cardiomyocytes Senescence via Regulating Redox State

Background

Cell senescence is central to a large body of age related pathology, and accordingly, cardiomyocytes senescence is involved in many age related cardiovascular diseases. In consideration of that, delaying cardiomyocytes senescence is of great importance to control clinical cardiovascular diseases. Previous study indicated that bradykinin (BK) protected endothelial cells from senescence induced by oxidative stress. However, the effects of bradykinin on cardiomyocytes senescence remain to be elucidated. In this study, we investigated the effect of bradykinin on H₂O₂-induced H9C2 cells senescence.

Methods and Results

Bradykinin pretreatment decreased the senescence induced by H₂O₂ in cultured H9C2 cells in a dose dependent manner. Interestingly, 1 nmol/L of BK almost completely inhibited the increase in senescent cell number and p21 expression induced by H₂O₂. Since H₂O₂ induces senescence through superoxide-induced DNA damage, we also observed the DNA damage by comet assay, and BK markedly reduced DNA damage induced by H₂O₂, and moreover, BK treatment significantly prevented reactive oxygen species (ROS) production in H9C2 cells treated with H₂O₂. Importantly, when co-incubated with bradykinin B2 receptor antagonist HOE-140 or eNOS inhibitor N-methyl-L-arginine acetate salt (L-NAME), the protective effects of bradykinin on H9C2 senescence were totally blocked. Furthermore, BK administration significantly prevented the increase in nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity characterized by increased ROS generation and gp91 expression and increased translocation of p47 and p67 to the membrane and the decrease in superoxide dismutase (SOD) activity and expression induced by H₂O₂ in H9C2 cells, which was dependent on BK B2 receptor mediated nitric oxide (NO) release.

Conclusions

Bradykinin, acting through BK B2 receptor induced NO release, upregulated antioxidant Cu/Zn-SOD and Mn-SOD activity and expression while downregulating NADPH oxidase activity and subsequently inhibited ROS production, and finally protected against cardiomyocytes senescence induced by oxidative stress.     

Overexpression of apolipoprotein J in human fibroblasts protects against cytotoxicity and premature senescence induced by ethanol and tert-butylhydroperoxide

Human diploid fibroblasts (HDFs) exposed to subcytotoxic stresses under H2O2, tert-butylhydroperoxide (t-BHP), and ethanol (EtOH) undergo stress-induced premature senescence (SIPS) characterized by many biomarkers of HDFs replicative senescence. Among these biomarkers are a growth arrest, an increase in the senescence-associated beta-galactosidase activity, a senescent morphology, an overexpression of p21waf-1 and the subsequent inability to phosphorylate pRb, the presence of the common 4977-bp mitochondrial deletion, and an increase in the steady-state level of several senescence-associated genes such as apolipoprotein J (apo J). Apo J has been described as a survival gene against cytotoxic stress. In order to study whether apo J would be protective against cytotoxicity SIPS and replicative senescence in human fibroblasts, a full-length complementary deoxyribonucleic acid of apo J was transfected into WI-38 HDFs and SV40-transformed WI-38 HDFs. The overexpression of apo J resulted in an increased cell survival after t-BHP and EtOH stresses at cytotoxic concentrations. In addition, when WI-38 HDFs were exposed to 5 subcytotoxic stresses with EtOH or t-BHP, in conditions that were previously shown to induce SIPS, a lower induction of 2 biomarkers of SIPS was observed in HDFs overexpressing apo J. No effect of apo J overexpression was observed on the proliferative life span of HDFs, even if apo J overexpression triggered osteonectin (SPARC) overexpression, which was shown to decrease the mitogenic potential of platelet-derived growth factor but not of other common growth-inducing conditions. Apo J senescence-related overexpression is proposed to have antiapoptotic rather than antiproliferative effects.     

BTG2 antagonizes Pin1 in response to mitogens and telomere disruption during replicative senescence

Cellular senescence limits the replicative capacity of normal cells and acts as an intrinsic barrier that protects against the development of cancer. Telomere shortening–induced replicative senescence is dependent on the ATM‐p53‐p21 pathway but additional genes likely contribute to senescence. Here, we show that the p53‐responsive gene BTG2 plays an essential role in replicative senescence. Similar to p53 and p21 depletion, BTG2 depletion in human fibroblasts leads to an extension of cellular lifespan, and ectopic BTG2 induces senescence independently of p53. The anti‐proliferative function of BTG2 during senescence involves its stabilization in response to telomere dysfunction followed by serum‐dependent binding and relocalization of the cell cycle regulator prolyl isomerase Pin1. Pin1 inhibition leads to senescence in late‐passage cells, and ectopic Pin1 expression rescues cells from BTG2‐induced senescence. The neutralization of Pin1 by BTG2 provides a critical mechanism to maintain senescent arrest in the presence of mitogenic signals in normal primary fibroblasts.     

Translocation of Ethanolamine Phosphoglyceride is Required for Initiation of Apoptotic Death in OLN-93 Oligodendroglial Cells

The possible interplay between extracellular signal-regulated protein kinase (ERK) activation and ethanolamine phosphoglycerides (PG) membrane bilayer translocation following oxidative stress (OS) (0.5 mM H₂O₂/0.05 mM Fe²⁺), was examined in oligodendroglia, OLN93, cells with altered plasma membrane PG composition. Cells supplemented with 50 μM docosahexaenoic acid (DHA, 22:6n3) to increase the number of potential double bond targets for OS in ethanolamine-PG (EPG) were compared to cells with diminished content of EPG, attained by the addition of 0.5 mM N,N-dimethylethanolamine (dEa). After 30 min OS, EPG translocation accompanied by sustained ERK activation and nuclear translocation culminating in apoptosis was found in DHA-supplemented cells in contrast to no EPG translocation, a brief ERK activation, but no nuclear translocation, and no cell death in DHA/dEa-supplemented cells. DHA/dEa-supplemented cells pretreated with the protein-tyrosine phosphatases inhibitor Na₃VO₄ followed by OS, although expressing a sustained ERK activation and nuclear translocation, failed to show apoptosis and lacked EPG translocation. In DHA-supplemented cells U0126, a MEK inhibitor, prevented ERK activation and EPG translocation and protected from cell death. These findings most likely indicate that ERK activation is an indispensable component for the signaling cascades leading to EPG translocation but only activation of the latter is leading to OS-induced apoptotic cell death.     

Senescence-related functional nuclear barrier by down-regulation of nucleo-cytoplasmic trafficking gene expression

One of the characteristic natures of senescent cells is the hypo- or irresponsiveness not only to growth factors but also to apoptotic stress. In the present study, we confirmed the inhibition of nuclear translocation of activated p-ERK1/2 and NF-kB p50 in response to growth stimuli or LPS in the senescent human diploid fibroblasts. In order to elucidate the underlying mechanism for the senescence-associated hypo-responsiveness, we carried out the comparison study for gene expression profiles through microarray analysis. In consequence, we observed the vast reduction in expression of nucleo-cytoplasmic trafficking genes in senescent cells, when compared with those in young cells. Expression levels of several nucleoporins, karyopherin α, karyopherin β, Ran, and Ran-regulating factors were confirmed to be down-regulated in senescent HDFs by using RT-PCR and Western blot methods. Taken together, these data suggest the operation of certain senescence-associated functional nuclear barriers by down-regulation of the nucleo-cytoplasmic trafficking genes in the senescent cells.     

Iduna protects HT22 cells from hydrogen peroxide-induced oxidative stress through interfering poly(ADP-ribose) polymerase-1-induced cell death (parthanatos)

Oxidative stress-induced cell death is common in many neurological diseases. However, the role of poly(ADP-ribose) polymerase-1-induced cell death (parthanatos) has not been fully elucidated. Here, we found that hydrogen peroxide (H₂O₂) could lead to PARP-1 activation and apoptosis-inducing factor nuclear translocation in a concentration dependent manner. Iduna, as a novel regulator of parthanatos, was also induced by H₂O₂. Down-regulation of Iduna by genetic ablation promoted H₂O₂-induced cell damage. Up-regulation of Iduna reduced the loss of mitochondrial potential and ATP and NAD + production, but did not affect the mitochondrial dysfunction-induced cytochrome c release, increase of Bax/Bcl-2 ratio, and Caspase-9/Caspase-3 activity. In contrast, overexpression of Iduna inhibited activation of PARP-1 and nuclear translocation of AIF. Further study showed that PARP-1 specific inhibitor, DPQ, blocked the protective effect of Iduna against H₂O₂-induced oxidative stress. Moreover, in the presence of proteasome inhibitor (MG-132) or ubiquitin E1 inhibitor (PYR-41), protective effect of Iduna was significantly weaken. These results indicate that Iduna acts as a potential antioxidant by improving mitochondrial function and inhibiting oxidative stress-induced parthanatos, and these protective effects are dependent on the involvement of ubiquitin–proteasome system.     

Reactive oxygen species control senescence‐associated matrix metalloproteinase‐1 through c‐Jun‐N‐terminal kinase

The lifetime exposure of organisms to oxidative stress influences many aging processes which involve the turnover of the extracellular matrix. In this study, we identify the redox‐responsive molecular signals that drive senescence‐associated (SA) matrix metalloproteinase‐1 (MMP‐1) expression. Precise biochemical monitoring revealed that senescent fibroblasts increase steady‐state (H₂O₂) 3.5‐fold (13.7–48.6 pM) relative to young cells. Restricting H₂O₂ production through low O₂ exposure or by antioxidant treatments prevented SA increases in MMP‐1 expression. The H₂O₂‐dependent control of SA MMP‐1 is attributed to sustained JNK activation and c‐jun recruitment to the MMP‐1 promoter. SA JNK activation corresponds to increases and decreases in the levels of its activating kinase (MKK‐4) and inhibitory phosphatase (MKP‐1), respectively. Enforced MKP‐1 expression negates SA increases in JNK phosphorylation and MMP‐1 production. Overall, these studies define redox‐sensitive signaling networks regulating SA MMP‐1 expression and link the free radical theory of aging to initiation of aberrant matrix turnover. J. Cell. Physiol. 225: 52–62, 2010. © 2010 Wiley‐Liss, Inc.