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 ß-galactosidase (SA-ß-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.

Protective effect of 4,4'-diaminodiphenylsulfone against paraquat-induced mouse lung injury.

Although 4,4'-diaminodiphenylsulfone (DDS, dapsone) has been used to treat several dermatologic conditions, including Hansen disease, for the past several decades, its mode of action has remained a topic of debate. We recently reported that DDS treatment significantly extends the lifespan of the nematode C. elegans by decreasing the generation of reactive oxygen species. Additionally, in in vitro experiments using non-phagocytic human fibroblasts, we found that DDS effectively counteracted the toxicity of paraquat (PQ). In the present study, we extended our work to test the protective effect of DDS against PQ in vivo using a mouse lung injury model. Oral administration of DDS to mice significantly attenuated the lung tissue damage caused by subsequent administration of PQ. Moreover, DDS reduced the local expression of mRNA transcripts encoding inflammation-related molecules, including endothelin-1 (ET-1), macrophage inflammatory protein-1α (MIP-1α), and transforming growth factor-ß (TGF-ß). In addition, DDS decreased the PQ-induced expression of NADPH oxidase mRNA and activation of protein kinase Cµ (PKCµ). DDS treatment also decreased the PQ-induced generation of superoxide anions in mouse lung fibroblasts. Taken together, these data suggest the novel efficacy of DDS as an effective protective agent against oxidative stress-induced tissue damages.

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 (H(2)O(2)), 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), H(2)O(2)-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 H(2)O(2) 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 H(2)O(2) 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 H(2)O(2) 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.

Restoration of senescent human diploid fibroblasts by modulation of the extracellular matrix.

Human diploid fibroblasts have the capacity to complete a finite number of cell divisions before entering a state of replicative senescence characterized by growth arrest, changes in morphology, and altered gene expression. Herein, we report that interaction with extracellular matrix (ECM) from young cells is sufficient to restore aged, senescent cells to an apparently youthful state. The identity of the restored cells as having been derived from senescent cells has been confirmed by a variety of methods, including time lapse live cell imaging and DNA finger print analysis. In addition to cell morphology, phenotypic restoration was assessed by resumption of proliferative potential, growth factor responsiveness, reduction of intracellular reactive oxygen species levels, recovery of mitochondrial membrane potential, and increased telomere length. Mechanistically, we find that both Ku and SIRT1 are induced during restoration and are required for senescent cells to return to a youthful phenotype. These observations demonstrate that human cellular senescence is profoundly influenced by cues from the ECM, and that senescent cell plasticity is much greater than that was previously believed to be the case.

Biliverdin reductase A in the prevention of cellular senescence against oxidative stress.

Biliverdin reductase A (BLVRA), an enzyme that converts biliverdin to bilirubin, has recently emerged as a key regulator of the cellular redox cycle. However, the role of BLVRA in the aging process remains unclear. To study the role of BLVRA in the aging process, we compared the stress responses of young and senescent human diploid fibroblasts (HDFs) to the reactive oxygen species (ROS) inducer, hydrogen peroxide (H2O2). H2O2 markedly induced BLVRA activity in young HDFs, but not in senescent HDFs. Additionally, depletion of BLVRA reduced the H2O2-dependent induction of heme oxygenase-1 (HO-1) in young HDFs, but not in senescent cells, suggesting an aging-dependent differential modulation of responses to oxidative stress. The role of BLVRA in the regulation of cellular senescence was confirmed when lentiviral RNAi- transfected stable primary HDFs with reduced BLVRA expression showed upregulation of the CDK inhibitor family members p16, p53, and p21, followed by cell cycle arrest in G0-G1 phase with high expression of senescence-associated ß-galactosidase. Taken together, these data support the notion that BLVRA contributes significantly to modulation of the aging process by adjusting the cellular oxidative status.

Reduction of Nup107 attenuates the growth factor signaling in the senescent cells.

Hypo-responsiveness to growth factors is a fundamental feature of cellular senescence. In this study, we found markedly decreased level of Nup107, a key scaffold protein in nuclear pore complex assembly, in senescent human diploid fibroblasts as well as in organs of aged mice. Depletion of Nup107 by specific siRNA in young human diploid fibroblasts prevented the effective nuclear translocation of phosphorylated extracellular signal-regulated kinase (ERK) following epidermal growth factor (EGF) stimulation, and decreased the expression of c-Fos in consequence. The disturbances in ERK signaling in Nup107 depleted cells closely mirror the similar changes in senescent cells. Knockdown of Nup107 in anaplastic oligodendroglioma cells caused cell death, rather than growth retardation, indicating a greater sensitivity to Nup107 depletion in cancer cells than in normal cells. These findings support the notion that Nup107 may contribute significantly to the regulation of cell fate in aged and transformed cells by modulating nuclear trafficking of signal molecules.

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 alpha, karyopherin beta, 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.

Age associated high level of major vault protein is p53 dependent.

Major vault protein (MVP) represents the main component of vaults and has been linked to multi-drug resistance (MDR) in cancer cells. We previously reported that MVP plays an important role in the resistance of senescent human diploid fibroblasts (HDFs) to apoptosis and also that MVP expression is markedly reduced in young HDFs but not in senescent HDFs. In this study, designed to elucidate the regulation of MVP in young and senescent HDFs, we examined the levels of transcriptional factors for the MVP gene, which revealed that among the putative transcriptional factors, p53 decreased only in young HDFs, but not in senescent HDFs in response to H(2)O(2) treatment in the same mode as the expression of MVP. Moreover, the phosphorylation status of p53 increased only in senescent HDFs but not in young HDFs in response to H(2)O(2) treatment. Therefore, we tested the possibility of MVP regulation by p53 status. MVP is upregulated in p53 over-expressing young HDFs, while MVP is downregulated in p53-specific small interfering RNA (siRNA)-transfected senescent HDFs, which suggests that the expression of MVP would be p53 dependent. Furthermore, using chromatin immunoprecipitation (ChIP) assay, we observed that p53 binds directly to the MVP promoter. Taken together, these results suggest that p53 would be a major transcriptional factor for MVP gene expression.