Proteomic analysis reveals novel common genes modulated in both replicative and stress-induced senescence.

Cellular senescence causes profound changes in gene expression profile. In this study, we used a combined 2D-DIGE and nanoLC-ESI-LIT-MS/MS approach to evaluate the proteomic changes occurring both in replicative and stress-induced senescence of human IMR90 cells. Twenty protein spots were identified as shifting their quantitative representation in the same direction (over- or down-represented) in both conditions of senescence, which were associated with 25 sequence entries. Dedicated experiments demonstrated that the decreased representation of a set of these proteins is associated with the down-regulation of the corresponding mRNAs, indicating that the regulation of these genes during the senescence process occurs at a transcriptional level. We also performed functional studies by silencing nine of these genes in young cells, which demonstrated that RNA interference-mediated knockdown of LEPRE1, LIMA1/EPLIN, MAGOHA and MAGOHB induces a premature senescent phenotype in IMR90 cells. Chromatin immunoprecipitation experiments indicated that the reduced expression of these four genes is associated with changes in the histone methylation pattern of their promoters, as proved by the occurrence of increased repressive H3K27me3 along with decreased active H3K4me3 marks, respectively. BIOLOGICAL SIGNIFICANCE: Cellular senescence, a stable form of cell cycle arrest, is recognized as a phenomenon related to aging and age-related pathologies as well as interfering with tumor progression. Gene expression changes are closely associated with the onset of senescence but the molecular pathways regulating this process are still poorly understood. By using proteomics coupled to functional studies, we here show that both replicative and stress-induced senescence share quantitative modification of four novel proteins, in addition to others already reported in the literature. When ectopically down-regulated, corresponding four genes induce a premature senescence in young cells. The observed parallelism concerning the down-regulation of these genes both in vitro and in vivo senescent cells may foresee a possible biomarker role of the corresponding proteins in monitoring the progression of both aging and age-related diseases. In conclusion, these results for the first time highlight a possible role of LEPRE1, LIMA1/EPLIN, MAGOHA and MAGOHB in the biology of cellular senescence/aging, thus contributing to gain a deeper knowledge of the molecular mechanisms involved in the senescence program.

Comparative analysis of gene expression data reveals novel targets of senescence-associated microRNAs.

In the last decades, cellular senescence is viewed as a complex mechanism involved in different processes, ranging from tumor suppression to induction of age-related degenerative alterations. Senescence-inducing stimuli are myriad and, recently, we and others have demonstrated the role exerted by microRNAs in the induction and maintenance of senescence, by the identification of a subset of Senescence-Associated microRNAs (SAmiRs) up-regulated during replicative or stress-induced senescence and able to induce a premature senescent phenotype when over-expressed in human primary cells. With the intent to find novel direct targets of two specific SAmiRs, SAmiR-494 and -486-5p, and cellular pathways which they are involved in, we performed a comparative analysis of gene expression profiles available in literature to select genes down-regulated upon replicative senescence of human primary fibroblasts. Among them, we searched for SAmiR's candidate targets by analyzing with different target prediction algorithms their 3'UTR for the presence of SAmiR-binding sites. The expression profiles of selected candidates have been validated on replicative and stress-induced senescence and the targeting of the 3'UTRs was assessed by luciferase assay. Results allowed us to identify Cell Division Cycle Associated 2 (CDCA2) and Inhibitor of DNA binding/differentiation type 4 (ID4) as novel targets of SAmiR-494 and SAmiR-486-5p, respectively. Furthermore, we demonstrated that the over-expression of CDCA2 in human primary fibroblasts was able to partially counteract etoposide-induced senescence by mitigating the activation of DNA Damage Response.

Identification of miR-494 direct targets involved in senescence of human diploid fibroblasts.

Cellular senescence is a permanent cell cycle arrest triggered by different stimuli. We recently identified up-regulation of microRNA (miR)-494 as a component of the genetic program leading to senescence of human diploid IMR90 fibroblasts. Here, we used 2-dimensional differential gel electrophoresis (2D-DIGE) coupled to mass spectrometry to profile protein expression changes induced by adoptive overexpression of miR-494 in IMR90 cells. miR-494 induced robust perturbation of the IMR90 proteome by significantly (P≤0.05) down-regulating a number of proteins. Combination of mass spectrometry-based identification of down-regulated proteins and bioinformatic prediction of the miR-494 binding sites on the relevant mRNAs identified 26 potential targets of miR-494. Among them, computational analysis identified 7 potential evolution-conserved miR-494 targets. Functional miR-494 binding sites were confirmed in 3'-untranslated regions (UTRs) of 4 of them [heterogeneous nuclear ribonucleoprotein A3 (hnRNPA3), protein disulfide isomerase A3 (PDIA3), UV excision repair protein RAD23 homolog B (RAD23B), and synaptotagmin-binding cytoplasmic RNA-interacting protein (SYNCRIP)/heterogeneous nuclear ribonucleoprotein Q (hnRNPQ)]. Their reduced expression correlated with miR-494 up-regulation in senescent cells. RNA interference-mediated knockdown of hnRNPA3 and, to a lesser extent, RAD23B mirrored the senescent phenotype induced by miR-494 overexpression, blunting cell proliferation and causing up-regulation of SA-ß-galactosidase and DNA damage. Ectopic expression of hnRNPA3 or RAD23B slowed the appearance of the senescent phenotype induced by miR-494. Overall, these findings identify novel miR-494 direct targets that are involved in cellular senescence.

p53 suppresses the Nrf2-dependent transcription of antioxidant response genes.

Cells respond to the shift of intracellular environment toward pro-oxidant conditions by activating the transcription of numerous "antioxidant" genes. This response is based on the activation of the Nrf2 transcription factor, which transactivates the genes containing in their promoters the antioxidant response cis-elements (AREs). If the oxidative stress provokes DNA damage, a second response of the cell takes place, based on the activation of p53, which induces cell cycle arrest and/or apoptosis. Here we have explored the cross-talk between these two regulatory mechanisms. The results show that p53 counteracts the Nrf2-induced transcription of three ARE-containing promoters of the x-CT, NQO1, and GST-alpha1 genes. Endogenous transcripts of these antioxidant genes accumulate as a consequence of Nrf2 overexpression or exposure to electrophile diethylmaleate, but these effects are again blocked by p53 overexpression or endogenous p53 activation. Chromatin immunoprecipitation experiments support the hypothesis that this p53-dependent trans-repression is due to the direct interaction of p53 with the ARE-containing promoters. Considering that p53-induced apoptosis requires an accumulation of reactive oxygen species, this negative control on the Nrf2 transactivation appears to be aimed to prevent the generation of a strong anti-oxidant intracellular environment that could hinder the induction of apoptosis.

Transcription regulation in NIH3T3 cell clones resistant to diethylmaleate-induced oxidative stress and apoptosis.

To investigate the molecular mechanisms underlying the induction of cell resistance to oxidative stress, NIH3T3 cell clones (NIH-DEM clones) were isolated and selected for their ability to survive the exposure to diethylmaleate (DEM), a glutathione-depleting agent. The oxidative stress-resistant phenotype of these clones is stable for at least 1 month in the absence of DEM, and includes the resistance also to other apoptosis-inducing stimuli. The expression profile of several antioxidant genes was examined in four of the DEM-resistant clones in the presence and in absence of DEM. The response to the acute exposure to DEM is similar in wild type and DEM-resistant cells, with the exception of the glutathione-S-transferase alpha1 gene, whose expression is highly induced in NIH-DEM clones. However, in the absence of an acute stress, the expression of some genes is higher in DEM-resistant clones than in wild-type cells and the gene expression profile significantly varies among the clones. In particular, glutathione-S-transferase alpha1 and cystine/glutamate transporter mRNAs are increased in NIH-DEM-12. In these cells, the promoters of the two genes drive a stronger transcription than in wild-type cells, and this appears to be dependent on the transcription factor Nrf2.

Fibromodulin gene transcription is induced by ultraviolet irradiation, and its regulation is impaired in senescent human fibroblasts.

Cells undergoing replicative senescence display an altered pattern of gene expression. Senescent fibroblasts show significant changes in the expression of mRNAs encoding extracellular matrix-remodeling proteins; among these mRNAs, the mRNA encoding fibromodulin is highly decreased in these cells. To understand the molecular basis of this phenomenon, we explored the regulatory mechanisms of the human fibromodulin gene. We found that fibromodulin gene promoter contains a cis-element, crucial for its basal expression, that forms a DNA-protein complex when exposed to nuclear extracts from exponentially growing human fibroblasts and not to extracts from cells undergoing senescence by repeated in vitro passages or by mild oxidative stress. The purification of this complex showed that it contains the damage-specific DNA-binding protein DDB-1. The latter is known to be induced by UV irradiation; therefore we checked whether fibromodulin gene promoter is regulated upon the exposure of the cells to UV rays. The results showed that, in exponentially growing fibroblasts, the promoter efficiency is increased by UV irradiation and the DDB-1-containing complex is robustly enriched in cells exposed to UV light. Accordingly, in these experimental conditions the endogenous fibromodulin mRNA accumulates to very high levels. On the contrary, senescent cells did not show any activation of the fibromodulin gene promoter, any induction of the DDB-1-containing complex, or any accumulation of fibromodulin mRNA. These phenomena are accompanied in senescent cells by a decrease of the UV-damaged DNA binding activity.

Redox control of signal transduction, gene expression and cellular senescence.

Reactive oxygen species (ROS) act as subcellular messengers in such complex cellular processes as mitogenic signal transduction, gene expression, regulation of cell proliferation, replicative senescence, and apoptosis. They serve to maintain cellular homeostasis and their production is under strict control. However, the mechanisms whereby ROS act are still obscure. Here we review recent advances in our understanding of signaling mechanisms and recent data about the involvement of ROS in: (i) the regulation of the mitogenic transduction elements, particularly protein kinases and phosphatases; (ii) the regulation of gene expression; and (iii) the induction of replicative senescence and the role, if any, in aging and age-related disorders.

Low-affinity receptor-mediated induction of superoxide by N-formyl-methionyl-leucyl-phenylalanine and WKYMVm in IMR90 human fibroblasts.

We investigated in IMR90 cells the effects of N-formyl-Met-Leu-Phe (N-fMLP) and WKYMVm (W peptide) on activation of the NADPH oxidase-like enzyme. In serum-deprived human fibroblasts, exposure to 100 microM N-fMLP or 10 microM peptide W for 1 min induced both p47phox translocation and NADPH-dependent superoxide generation. These effects were in large part mediated by prevention of the rapid activation of extracellular signal-regulated kinases (ERKs) by preincubation with the MEK1 inhibitor PD098059. Furthermore, responses to N-fMLP or W peptide were inhibited by pertussis toxin, suggesting the involvement of a seven-transmembrane G protein-coupled receptor(s) for peptides. RT-PCR experiments demonstrated the expression in these cells of the low-affinity receptor FPRL1, but not the high-affinity receptor FPR. Incubation with radiolabeled WKYMVm, which had a higher efficiency on FPRL1, revealed that human fibroblasts express binding sites for 125I-WKYMVm that are specifically displaced by increasing concentrations of unlabeled ligand. Analysis of the binding data predicted a Kd of 155.99 nM and a receptor density of about 16,200 molecules/cell. HEK293 cells, which express a NADPH oxidase-like enzyme but not formyl peptide receptors, transiently transfected with FPRL1 cDNA produced superoxide on stimulation with N-fMLP or W peptide, demonstrating that this receptor is biologically functional.

Protein kinase B activation by reactive oxygen species is independent of tyrosine kinase receptor phosphorylation and requires SRC activity.

Reactive oxygen species (ROS) participate as second messengers in the mitogenic signal transduction. Most of the experimental data supporting the role of ROS as signaling molecules have been obtained by using H2O2. Exposure of cells to H2O2 rapidly increases tyrosine phosphorylation of tyrosine kinase receptors (TKRs) in the absence of growth factor binding, thus inducing the activation of downstream signaling cascades, like that of protein kinase B (AKT). Another molecule able to induce an increase of intracellular ROS levels is diethylmaleate (DEM), which acts by depleting the ROS scavenger reduced glutathione (GSH). A comparison of the effects exerted by H2O2 and DEM shows that the latter induces redox modifications milder than those generated by H2O2. We also demonstrated that DEM-induced redox modifications are not accompanied by platelet-derived growth factor-receptor (PDGF-R) and epidermal growth factor-receptor Tyr phosphorylation, although they are able to activate ERKs and AKT, with kinetics different from those observed following H2O2 treatment. The activation of these two pathways is not blocked by AG1296, a selective inhibitor of PDGF-R Tyr kinase, thus confirming that the effects of DEM are not mediated by the TKR phosphorylation. On the contrary, PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazole[3,4-d]pyrimidine), an inhibitor of Src kinase, completely prevents DEM- and H2O2-induced AKT activation but has no effect on the pathway of ERKs. Finally, nitration of Tyr residues in PDGF-R is observed in DEM-treated cells, thus suggesting that ROS-induced modifications different from Tyr phosphorylation can occur at the growth factor-receptor level and can be involved in the regulation of signaling pathways.

An alternative model of H ferritin promoter transactivation by c-Jun.

c-Jun is a member of the activator protein 1 family, and its interaction with different nuclear factors generates a wide spectrum of complexes that regulate transcription of different promoters. H ferritin promoter transcription is tightly dependent on nuclear factor Y (NFY). Ferritin transcription is activated by c-Jun, although the promoter does not contain a canonical binding site. NFY, on the other hand, does not bind c-Jun in vitro, whereas in vivo c-Jun is found in the complex containing NFY. Moreover, a c-Jun-GCN4 chimaeric construct containing only the transactivation domain of Jun and the basic-region leucine-zipper domain of GCN4 stimulates the H ferritin promoter. A synthetic GAL4 promoter and the cognate activator, the fusion protein NFY-GAL4, are potently activated by c-Jun. Titration of p300 by co-expressing E1A abolishes the stimulatory effect. Moreover, another p300-dependent promoter, the cAMP-response element, can be superactivated by c-Jun using the same mechanism. These data indicate that c-Jun, when activated or overexpressed, is recruited to the H ferritin promoter by p300, which links NFY, bound to DNA, to the complex. These results add a new level of complexity to transcriptional regulation by c-Jun, which can activate p300-dependent promoters without binding directly to the target DNA.

Inhibition of NADH/NADPH oxidase affects signal transduction by growth factor receptors in normal fibroblasts.

Reactive oxygen species have been implicated as possible second messengers in mitogenic signal transduction. We demonstrate that in normal fibroblasts the treatment with the two inhibitors of phagocytic NADH/NADPH oxidase prevents tyrosine phosphorylation of platelet-derived growth factor receptor upon the exposure of serum-deprived cells to growth factors. Furthermore, the inhibition of NADH/NADPH oxidase abolishes ERKs activation and p21(waf1) accumulation that occurs when cells are exposed to growth factors. Finally, NADH/NADPH inhibitors prevent the p66(Shc) Ser-phosphorylation induced by serum and by phorbol 12-myristate-13-acetate, which suggests that the direct target(s) of reactive oxygen species is(are) located upstream from the machinery connecting growth factor receptors to Ras.