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Background: Cystic fibrosis trans-membrane conductance regulator (CFTR) is a cAMP-modulated chloride/bicarbonate channel with different functions in various organs and tissues. Patients with CF are characterized by recurrent infections and by chronic neutrophilic airway inflammation as well as increased levels of neutrophil elastase (NE) in the airways. NE triggers cell cycle arrest that can lead to senescence. Moreover, it is well demonstrated that the CFTR expression get decreasing from embryogenesis to adult/elder age. Transcriptional, posttranscriptional and epigenetic regulatory mechanisms play a key role in decreasing the CFTR expression during life and recent evidences show that some CFTR-regulating microRNAs, i.e, mir-494, is up regulated during cell senescence. Cellular senescence is a permanent cell cycle arrest. In young cells it can be induced by different stimuli both exogenous and endogenous such as the physiological shortening of telomeres, the activation of oncogenes as well as oxidative stress. Furthermore, senescent cells are associated with modifications in the gene expression profile based on transcriptional, posttranscriptional and epigenetic regulatory mechanisms. Aims: To assess whether CFTR might be involved in cellular senescence and understand its role in the cellular senescence. Methods: To induce premature senescence, IMR90 cells at PDL34 were treated with 100 M diethylmaleate (DEM) (Sigma-Aldrich) in complete medium on alternate days for ten days. Western Blot and Real Time-PCR analysis were performed to assess either the CFTR and the microRNAs expression in: i) IMR90 cells at different population doubling levels (PDL); ii) IMR90 cells at different days after DEM treatment. Results:We found that: i) the CFTR expressions significantly decreased in IMR90 cells during replicative senescence (PDL58 versus PDL35); ii) the CFTR expression significantly decreased in IMR90 cells after DEM treatment; iii) there was an inverse relationship between CFTR and miRNA 494 expression either in IMR90 cells during replicative senescence and in IMR90 cells after DEM treatment. Conclusions: Our preliminary data show that CFTR is physiologically down-regulated during cellular senescence and this may (at least partially) depend on the up-regulation of miR-494. To better investigate the role of CFTR in cellular senescence, we are performing the over-expression of CFTR protein in pre-senescent/senescent IMR90 and the silencing of CFTR in IMR90 young cells. Furthermore, we are now validating such results on primary epithelial nasal cells from different subjects.

We recently identified miR-494 up-regulation as a component of the program leading to cellular senescence of human diploid IMR90 fibroblasts [1]. To identify miR-494 targets, we used 2D-DIGE coupled to mass spectrometry analysis to profile protein changes induced by overexpression of miR-494 in IMR90 cells. miR-494 induced robust perturbation of the IMR90 proteome by significantly (p≤0.05) affecting a number of proteins. Combination of mass spectrometry-based identification of down-regulated proteins and bioinformatic prediction analysis for miR-494 binding sites on the relevant mRNAs, identified 26 putative targets of miR-494, with 7 of them featuring evolutionary conservation of miR-494 binding site. Functional miR-494 binding site were confirmed for hnRNPA3, PDIA3, RAD23B, and SYNCRIP. siRNA-mediated knockdown of hnRNPA3 and, to a lesser extent, RAD23B mirrored the senescent phenotype induced by miR-494, blunting cell proliferation and causing increase of SA-β-gal and DNA damage. Reintroduction of hnRNPA3 or RAD23B slowed the appearance of miR-494-induced senescent phenotype in IMR90 cells. Overall, these findings identify novel miR-494 direct targets involved in senescence. References: [1]Faraonio R et al. Cell Death Differ. 2012 Apr;19(4):713- 21. doi: 10.1038/cdd.2011.143. Epub 2011 Nov 4

Cellular senescence is a permanent cell cycle arrest triggered by different stimuli such as telomere attrition, activated oncogenes, oxidative stress. We recently identified up-regulation of miR-494 as a component of the genetic program leading to senescence of human diploid IMR90 fibroblasts. Here, we used 2D-DIGE coupled to mass spectrometry analysis 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 analysis of the miR-494 binding sites on the relevant mRNAs identified 26 potential targets of miR-494. Among them, computational target prediction analysis identified 7 putative miR-494 targets, the 3’UTRs of which featured evolutionary conservation of binding sites for miR-494. Functional miR-494 binding sites were confirmed in 3’UTRs of four of them (hnRNPA3, PDIA3, RAD23B, and SYNCRIP/hnRNPQ). Accordingly, their reduced expression correlated with miR-494 up-regulation in different models of senescence. 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-β-galactosidase and DNA damage. Ectopic expression of hnRNPA3 or RAD23B slowed the appearance of the senescent phenotype induced by miR-494 in IMR90 cells. Overall, these findings identify novel miR-494 direct targets that are involved in cellular senescence.

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.

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.

A cDNA minilibrary in pUC18 has been generated from 3-month-old rat brains. Two hundred clones were randomly selected and sequenced. Comparison with a nucleic acid and protein data bank revealed a number of cDNA fragments not homologous to any published sequence. Northern blot analyses of some of these clones yielded 5 brain-specific cDNA fragments. The full-length cDNA for one of these clones has been isolated and completely sequenced. It corresponds to an mRNA of about 1,500 nucleotides, which is present in brain and, to a lesser extent, in heart and skeletal muscle. Its expression is developmentally regulated in the rat brain from 14-day embryos to 3-month-old adults. A very recent comparison with the EMBL nucleotide sequence data bank showed that this mRNA codes for a brain-specific snRNP-associated protein.