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Supplementary Figure Legends 1-4 from Cathepsin D and Eukaryotic Translation Elongation Factor 1 as Promising Markers of Cellular Senescence

Supplementary Table 2 from Cathepsin D and Eukaryotic Translation Elongation Factor 1 as Promising Markers of Cellular Senescence

Supplementary Figure 2 from Cathepsin D and Eukaryotic Translation Elongation Factor 1 as Promising Markers of Cellular Senescence

Supplementary Figure 1 from Cathepsin D and Eukaryotic Translation Elongation Factor 1 as Promising Markers of Cellular Senescence

Supplementary Figure 3 from Cathepsin D and Eukaryotic Translation Elongation Factor 1 as Promising Markers of Cellular Senescence

Supplementary Figure 4 from Cathepsin D and Eukaryotic Translation Elongation Factor 1 as Promising Markers of Cellular Senescence

Supplementary Table 1 from Cathepsin D and Eukaryotic Translation Elongation Factor 1 as Promising Markers of Cellular Senescence

Summary Behçet's disease (BD) is a chronic systemic inflammatory disease with unclear etiopathogenesis. Although gene variants of CC chemokine receptor type 1 (CCR1) have been reported, the protein expression of CCR1 in patients with BD remains unclear. The objective of this study was to analyze the frequencies of CCR1+ cells in a herpes simplex virus‐induced mouse model of BD. The frequencies of CCR1+ cells on the surface and in the cytoplasm of peripheral blood mononuclear cells and lymph nodes were analyzed by flow cytometry. The CCR1+ cells were significantly down‐regulated in BD mice compared with the normal control and symptom‐free control mice. Colchicine and pentoxifylline treatment improved the symptoms of BD and increased the frequencies of CCR1+ cells in BD mice. Treatment with chemokine CC motif ligand 3 (CCL3), a ligand of CCR1, caused BD symptoms to deteriorate in 10 of 16 BD mice (62·5%) via down‐regulation of CCR1+ cells. Anti‐CCL3 antibody treatment ameliorated BD symptoms in 10 of 20 mice (50%) and significantly decreased the disease severity score compared with CCL3‐treated BD mice (P = 0·01) via up‐regulation of CCR1+ cell frequencies. In patients with BD, plasma levels of CCL3 in an active state were significantly higher than in healthy control individuals (P = 0·02). These results show that the up‐regulation of CCR1+ cells was related to the control of systemic inflammation of BD in mouse models., The frequencies of CCR1 positive cells were down‐regulated in peripheral blood leukocytes of Behçet's disease symptomatic mice. CCL3, a ligand of CCR1, induced down‐regulation of CCR1 positive cells. The treatment of anti‐CCL3 antibody up‐regulated CCR1 positive cells and had an improvement effect on inflammatory symptoms in Behçet's disease mice.

As senescence develops, cells sequentially acquire diverse senescent phenotypes along with simultaneous multistage gene reprogramming. It remains unclear what acts as the key regulator of the collective changes in gene expression at initiation of senescent reprogramming. Here we analyzed time series gene expression profiles obtained in two different senescence models in human diploid fibroblasts: replicative senescence and H2O2-induced senescence. Our results demonstrate that suppression of DNA methyltransferase 1 (DNMT1)-mediated DNA methylation activity was an initial event prior to the display of senescent phenotypes. We identified seven DNMT1-interacting proteins, ubiquitin-like with PHD and ring finger domains 1 (UHRF1), EZH2, CHEK1, SUV39H1, CBX5, PARP1, and HELLS (also known as LSH (lymphoid-specific helicase) 1), as being commonly down-regulated at the same time point as DNMT1 in both senescence models. Knockdown experiments revealed that, among the DNMT1-interacting proteins, only UHRF1 knockdown suppressed DNMT1 transcription. However, UHRF1 overexpression alone did not induce DNMT1 expression, indicating that UHRF1 was essential but not sufficient for DNMT1 transcription. Although UHRF1 knockdown effectively induced senescence, this was significantly attenuated by DNMT1 overexpression, clearly implicating the UHRF1/DNMT1 axis in senescence. Bioinformatics analysis further identified WNT5A as a downstream effector of UHRF1/DNMT1-mediated senescence. Senescence-associated hypomethylation was found at base pairs −1569 to −1363 from the transcription start site of the WNT5A gene in senescent human diploid fibroblasts. As expected, WNT5A overexpression induced senescent phenotypes. Overall, our results indicate that decreased UHRF1 expression is a key initial event in the suppression of DNMT1-mediated DNA methylation and in the consequent induction of senescence via increasing WNT5A expression.

Cellular senescence is a process by which cells enter a state of permanent cell cycle arrest. It is commonly believed to underlie organismal aging and age-associated diseases. However, the mechanism by which cellular senescence contributes to aging and age-associated pathologies remains unclear. Recent studies showed that senescent cells exert detrimental effects on the tissue microenvironment, generating pathological facilitators or aggravators. The most significant environmental effector resulting from senescent cells is the senescence-associated secretory phenotype (SASP), which is constituted by a strikingly increased expression and secretion of diverse pro-inflammatory cytokines. Careful investigation into the components of SASPs and their mechanism of action, may improve our understanding of the pathological backgrounds of age-associated diseases. In this review, we focus on the differential expression of SASP-related genes, in addition to SASP components, during the progress of senescence. We also provide a perspective on the possible action mechanisms of SASP components, and potential contributions of SASP-expressing senescent cells, to age-associated pathologies. [BMB Reports 2015; 48(10): 549-558]

Behçet’s disease (BD) is an autoinflammatory disease that can lead to life- and sight-threating complications. Dendritic cells (DCs) are the most potent antigen-presenting cells that can regulate multiple inflammatory pathways. The objective of this study was to investigate the association of the DC stimulatory molecule CD83 with BD. Frequencies of costimulatory molecules expressing DCs in peripheral blood leukocytes (PBL) were measured by flow cytometry (FACS). The severity of symptoms in HSV-1-induced BD symptomatic mice was also assessed. Frequencies of CD83-positive cells were significantly increased in mice exhibiting BD symptoms, compared to those in asymptomatic mice. Abatacept, a CD80/86 blocker, significantly decreased the frequencies of CD83-positive cells in a time- and dose-dependent manner. BD symptomatic mice treated with Abatacept showed gradual reduction in the severity score of symptoms. Intraperitoneal injection of CD83 siRNA significantly reduced the frequencies of CD83-positive cells in PBL and peritoneal macrophages. After CD83 siRNA injection, BD symptoms of mice were improved and disease severity was decreased. Discontinuation of CD83 siRNA deteriorated symptoms while readministration of CD83 siRNA again improved BD symptoms of mice. These results clearly indicate the involvement of CD83-expressing cells in the inflammatory symptoms of BD. Therefore, CD83 might be useful as a therapeutic target for BD.

The purpose of this study was to clarify the correlation between microRNA-21 (miR-21) expression and inflammation in a herpes simplex virus (HSV)-induced Behçet’s Disease (BD) mouse model. miR-21 was compared between BD patients and healthy controls in peripheral blood mononuclear cells (PBMC). For miR-21 inhibition, miR-21 antagomir was applied to BD mice. The change of symptoms was monitored. The levels of cytokines and related molecules were determined by ELISA and real time qPCR. Treatment with colchicine or pentoxifylline down-regulated the level of miR-21 with improved symptoms in mice. miR-21 inhibition was accompanied by down-regulated serum levels of IL-17 and IL-6. The expression levels of PDCD4, RhoB, PD-1, IL-12p35, and toll-like receptor-4 were also regulated by miR-21 inhibition. miR-21 was correlated with HSV-induced BD-like inflammation in mice and BD patients. The expression of miR-21 was regulated by antagomir in mice.

Osteoblasts are specialized mesenchymal cells that are responsible for bone formation. In this study, we examine the role of GATA4 in osteoblast differentiation. GATA4 was abundantly expressed in preosteoblast cells and gradually down-regulated during osteoblast differentiation. Overexpression of GATA4 in osteoblastic cells inhibited alkaline phosphatase activity and nodule formation in osteogenic conditioned cell culture system. In addition, overexpression of GATA4 attenuated expression of osteogenic marker genes, including Runx2, alkaline phosphatase, bone sialoprotein, and osteocalcin, all of which are important for osteoblast differentiation and function. Overexpression of GATA4 attenuated Runx2 promoter activity, whereas silencing of GATA4 increased Runx2 induction. We found that GATA4 interacted with Dlx5 and subsequently decreased Dlx5 binding activity to Runx2 promoter region. Our data suggest that GATA4 acts as a negative regulator in osteoblast differentiation by downregulation of Runx2. [BMB Reports 2014; 47(8): 463-468]

Transforming growth factor β1 (TGF β1) induces Mv1Lu cell senescence by persistently producing mitochondrial reactive oxygen species (ROS) through decreased complex IV activity. Here, we investigated the molecular mechanism underlying the effect of TGF β1 on mitochondrial complex IV activity. TGF β1 progressively phosphorylated the negative regulatory sites of both glycogen synthase kinase 3 (GSK3) α and β, corresponding well to the intracellular ROS generation profile. Pre-treatment of N -acetyl cysteine, an antioxidant, did not alter this GSK3 phosphorylation (inactivation), whereas pharmacological inhibition of GSK3 by SB415286 significantly increased mitochondrial ROS, implying that GSK3 phosphorylation is an upstream event of the ROS generation. GSK3 inhibition by SB415286 decreased complex IV activity and cellular O 2 consumption rate and eventually induced senescence of Mv1Lu cell. Similar results were obtained with siRNA-mediated knockdown of GSK3. Moreover, we found that GSK3 not only exists in cytosol but also in mitochondria of Mv1Lu cell and the mitochondrial GSK3 binds complex IV subunit 6b which has no electron carrier and is topologically located in the mitochondrial intermembrane space. Involvement of subunit 6b in controlling complex IV activity and overall respiration rate was proved with siRNA-mediated knockdown of subunit 6b. Finally, TGF β1 treatment decreased the binding of the subunit 6b to GSK3 and subunit 6b phosphorylation. Taken together, our results suggest that GSK3 inactivation is importantly involved in TGF β1-induced complex IV defects through decreasing phosphorylation of the subunit 6b, thereby contributing to senescence-associated mitochondrial ROS generation.

Increased cell mass is one of the characteristics of senescent cells, but this event has not been clearly defined. When subcellular organellar mass was estimated with organelle-specific fluorescence dyes, we observed that most membranous organelles progressively increase in mass during cell senescence. This increase was accompanied by an increase in membrane lipids and augmented expression of lipogenic enzymes, such as fatty acid synthase (FAS), ATP citrate lyase, and acetyl-CoA carboxylase. The mature form of sterol regulatory element-binding protein (SREBP)-1 was also elevated. Increased expression of these lipogenic effectors was further observed in the liver tissues of aging Fischer 344 rats. Ectopic expression of mature form of SREBP-1 in both Chang cells and primary young human diploid fibroblasts was enough to induce senescence. Blocking lipogenesis with FAS inhibitors (cerulenin and C75) and via siRNA-mediated silencing of SREBP-1 and ATP citrate lyase significantly attenuated H(2)O(2)-induced senescence. Finally, old human diploid fibroblasts were effectively reversed to young-like cells by challenging with FAS inhibitors. Our results suggest that enhanced lipogenesis is not only a common event, but also critically involved in senescence via SREBP-1 induction, thereby contributing to the increase in organelle mass (as a part of cell mass), a novel indicator of senescence.

Mitochondrial complex II defect has recently been implicated in cellular senescence and in the ageing process of which a critical phenotype is retardation and arrest of cellular growth. However, the underlying mechanisms of how complex II defect affects cellular growth, remain unclear. In this study, we investigated the effect of complex II inhibition using a subcytotoxic dose (400 microM) of 2-thenoyltrifluoroacetone (TTFA), a conventional complex II inhibitor, on cell cycle progression. TTFA (400 microM) directly decreased KCN-sensitive cellular respiration rate to 67% of control and disrupted the mitochondrial membrane potential. In contrast to other respiratory inhibitors such as rotenone, antimycin A, and oligomycin, TTFA prolonged the duration of each phase of the cell cycle (G1, S, and G2/M) equally, thereby delaying overall cell cycle progression. This delay was accompanied by a biphasic increase of reactive oxygen species (ROS) and concurrent glutathione oxidation, in addition to a slight decrease in the cellular ATP level. Finally, the delay in cell cycle progression caused by TTFA was proved to be mainly due to ROS overproduction and subsequent oxidative stress, as evidenced by its reversal following pretreatment with antioxidants. Taken together, these results suggest that an overall delay in cell cycle progression due to complex II defects may contribute to ageing and degenerative diseases via inhibition of cellular growth and proliferation without arrest at any specific phase of the cell cycle.

Although mitochondrial dysfunction has been implicated in tumor metastasis, it is unclear how it regulates tumor cell aggressiveness. We have reported previously that human hepatoma cells harboring mitochondrial defects have high tumor cell invasion activity via increased claudin-1 (Cln-1) expression. In this study, we demonstrated that mitochondrial respiratory defects induced Cln-1 transcription via reactive oxygen species (ROS)-mediated heat shock factor 1 (HSF1) activation, which contributed to hepatoma invasiveness. We first confirmed the inverse relationship between mitochondrial defects and Cln-1 induction in SNU hepatoma cells and hepatocellular carcinoma tissues. We then examined five different respiratory complex inhibitors, and complex I inhibition by rotenone most effectively induced Cln-1 at the transcriptional level. Rotenone increased both mitochondrial and cytosolic ROS. In addition, rotenone-induced Cln-1 expression was attenuated by N-acetylcysteine, an antioxidant, and exogenous H2O2 treatment was enough to increase Cln-1 transcription, implying the involvement of ROS. Next we found that ROS-mediated HSF1 activation via hyperphosphorylation was the key event for Cln-1 transcription. Moreover, the Cln-1 promoter region (from −529 to +53) possesses several HSF1 binding elements, and this region showed increased promoter activity and HSF1 binding affinity in response to rotenone treatment. Finally, we demonstrated that the invasion activity of SNU449 cells, which harbor mitochondrial defects, was blocked by siRNA-mediated HSF1 knockdown. Taken together, these results indicate that mitochondrial respiratory defects enhance Cln-1-mediated hepatoma cell invasiveness via mitochondrial ROS-mediated HSF1 activation, presenting a potential role for HSF1 as a novel mitochondrial retrograde signal-responsive transcription factor to control hepatoma cell invasiveness.

We reported a ratiometric two-photon fluorescent probe (SG1) for β-galactosidase (β-gal) and its application to quantitative detection of β-gal activity during cellular senescence in live cells and in aged tissues. This probe is characterized by a significant two-photon excited fluorescence, a marked blue-to-yellow emission color change in response to β-gal, easy loading, insensitivity to pH and reactive oxygen species (ROS), high photostability, and low cytotoxicity. In addition, we show that SG1 labeling is an effective tool for quantitative detection of senescence-associated β-gal activity at the subcellular level in situ. This finding demonstrates that SG1 will find useful applications in biomedical research, including studies of cell aging.

Transforming growth factor β1 (TGF-β1) induces Mv1Lu cell senescence through inactivating glycogen synthase kinase 3 (GSK3), thereby inactivating complex IV and increasing intracellular ROS. In the present study, we identified protein kinase C delta (PKCδ) as an upstream regulator of GSK3 inactivation in this mechanism of TGF-β1-induced senescence. When Mv1Lu cells were exposed to TGF-β1, PKCδ phosphorylation simultaneously increased with GSK3 phosphorylation, and then AKT and ERK were phosphorylated. AKT phosphorylation and Smad signaling were independent of GSK3 phosphorylation, but ERK phosphorylation was downstream of GSK3 inactivation. TGF-β1-triggered GSK3 phosphorylation was blocked by inhibition of PKCδ, using its pharmacological inhibitor, Rottlerin, or overexpression of a dominant negative PKCδ mutant, but GSK3 inhibition with SB415286 did not alter PKCδ phosphorylation. Activation of PKCδ by PMA delayed cell growth and increased intracellular ROS level, but did not induce senescent phenotypes. In addition, overexpression of wild type or a constitutively active PKCδ mutant was enough to delay cell growth and decrease the mitochondrial oxygen consumption rate and complex IV activity, but weakly induce senescence. However, PMA treatment on Mv1Lu cells, which overexpress wild type and constitutively active PKCδ mutants, effectively induced senescence. These results indicate that PKCδ plays a key role in TGF-β1-induced senescence of Mv1Lu cells through the phosphorylation of GSK3, thereby triggering mitochondrial complex IV dysfunction and intracellular ROS generation.

Summary Although senescence has long been implicated in aging-associated pathologies, it is not clearly understood how senescent cells are linked to these diseases. To address this knowledge gap, we profiled cellular senescence phenotypes and mRNA expression patterns during replicative senescence in human diploid fibroblasts. We identified a sequential order of gain-of-senescence phenotypes: low levels of reactive oxygen species, cell mass/size increases with delayed cell growth, high levels of reactive oxygen species with increases in senescence-associated β-galactosidase activity (SA-β-gal), and high levels of SA-β-gal activity. Gene expression profiling revealed four distinct modules in which genes were prominently expressed at certain stages of senescence, allowing us to divide the process into four stages: early, middle, advanced, and very advanced. Interestingly, the gene expression modules governing each stage supported the development of the associated senescence phenotypes. Senescence-associated secretory phenotype–related genes also displayed a stage-specific expression pattern with three unique features during senescence: differential expression of interleukin isoforms, differential expression of interleukins and their receptors, and differential expression of matrix metalloproteinases and their inhibitory proteins. We validated these phenomena at the protein level using human diploid fibroblasts and aging Sprague-Dawley rat skin tissues. Finally, disease-association analysis of the modular genes also revealed stage-specific patterns. Taken together, our results reflect a detailed process of cellular senescence and provide diverse genome-wide information of cellular backgrounds for senescence.

Senescence has been suggested as a defense mechanism to block sporadic induction of cancer cells. Radiation treatment induces proliferating cancer cells to turn into non-proliferating senescent cells in vitro. To characterize transcriptional reprogramming after radiation treatment, we measured the gene expression profiles of MCF7 at different time points after treatment. In these experiments, we found that IR induced premature senescence in MCF7 cells, and IR treatment resulted in significant changes in the expression of 305 marker genes (1% FDR), which were strongly correlated (|r|0.9) with IR treatment in a time-dependent manner. Functional analysis of these markers indicated that the dynamics of cytoskeletal structure and lysosomal activity gradually increased. The expression of maker genes for modulator proteins, that were responsible for the dynamics of actin stress fibers and focal adhesion, displayed a particularly strong positive correlation with senescence-associated (SA) morphological changes through time. We also observed a strong induction of genes related to lysosomal metabolic activity, which was accompanied by an increase in the number of SA-beta-Gal positive cells. However, the expression of genes for cell cycle progression, post-transcription and translation activities gradually decreased after radiation treatment. Especially, we observed clear cell cycle arrest specifically at the S and G2/M phases with consistent down-regulation of genes for microtubule assembly/disassembly or spindle biogenesis.

Induction of premature senescence may be a promising strategy for cancer treatment. However, biomarkers for senescent cancer cells are lacking. To identify such biomarkers, we performed comparative proteomic analysis of MCF7 human breast cancer cells undergoing cellular senescence in response to ionizing radiation (IR). IR-induced senescence was associated with up-regulation of cathepsin D (CD) and down-regulation of eukaryotic translation elongation factor 1β2 (eEF1B2), as confirmed by Western blot. The other elongation factor, eukaryotic translation elongation factor 1α1 (eEF1A1), was also down-regulated. IR-induced senescence was associated with similar changes of CD and eEF1 (eEF1A1 and eEF1B2) levels in the HCT116 colon cancer cell line and the H460 lung cancer cell line. Up-regulation of CD and down-regulation of eEF1 seemed to be specific to senescence, as they were observed during cellular senescence induced by hydrogen peroxide or anticancer drugs (camptothecin, etoposide, or 50 ng doxorubicin) but not during apoptosis induced by Taxol or 10 μg doxorubicin or autophagy induced by tamoxifen. The same alterations in CD and eEF1A1 levels were observed during replicative senescence and Ras oncogene-induced senescence. Transient cell cycle arrest did not alter levels of eEF1 or CD. Chemical inhibition of CD (pepstatin A) and small interfering RNA–mediated knockdown of CD and eEF1 revealed that these factors participate in cell proliferation. Finally, the senescence-associated alteration in CD and eEF1 levels observed in cell lines was also observed in IR-exposed xenografted tumors. These findings show that CD and eEF1 are promising markers for the detection of cellular senescence induced by a variety of treatments. [Cancer Res 2009;69(11):4638–47]

It is known that the level of interleukin-6 (IL-6) is higher in patients with active Behcet's disease (BD) than in those with inactive disease. Herpes simplex virus (HSV) type 1 inoculation of the earlobes of ICR mice resulted in the development of BD-like symptoms. To find out whether downregulation of IL-6 would affect the symptoms of BD, IL-6 small interfering RNA (siRNA) was administered to a BD mouse model. IL-6 siRNA was intraperitoneally injected into BD mice to downregulate IL-6 (n=9). IL-6 siRNA injection downregulated serum IL-6 level (118.9+/-114.4 pg ml(-1)) compared with scramble injection (439.4+/-378.0 pg ml(-1)) in BD mice (P=0.01). In seven out of nine IL-6 siRNA-injected BD mice, 77.8% improved and the severity score was decreased from 3.1+/-1.05 to 1.7+/-0.87 (P=0.005), whereas two out of six (33.3%) scramble-injected BD mice improved and the severity score changed from 2.5+/-0.84 to 2.0+/-1.41 (P=0.203). Foxp3, ROR gamma t, IL-17A, IL-17F and tumor necrosis factor-alpha were also influenced in IL-6 siRNA-injected BD mice compared with scramble-injected BD mice. Adoptive transfer of CD4+CD25+ cells to BD mice affected the decrease of IL-6 serum levels and were dependent on CD4+CD25+ cell numbers. These results showed that downregulation of IL-6 improved the inflammatory symptoms in BD mice through upregulation of regulatory T cells and inhibition of Th17 cells.

Mitochondria play a pivotal role as an ATP generator in aerobically growing cells, and their defects have long been implicated in the cellular aging process, although its detailed underlying mechanisms remain unclear. Recently, we found that, in the cellular senescent process of Chang cells induced by desferroxamine mesylate, an iron chelator, a significant decrease of intracellular ATP level was accompanied by decline in complex II activity, which preceded acquisition of the senescent phenotype. In the present study, we investigated the mechanism of how the mitochondrial ATP productivity was damaged by iron chelation and how complex II defect was involved in the senescent arrest. The ATP loss was irreversible and accompanied by sustained collapse of mitochondrial membrane potential (Delta psi m), but the ATP loss itself did not seem to be essential in progression to the senescent arrest. The Delta psi m disruption was due to decreased mitochondrial respiration, which was primarily associated with the defective complex II activity. Furthermore, we found that the declined activity of complex II was mainly due to down-regulation of protein expression of the iron-sulfur subunit, which was associated with the irreversibility of the arrest. Finally, we demonstrated that specific inhibition of complex II with 2-thenoyltrifluoroacetone induced overall delay of the cell cycle, suggesting that the delayed arrest by desferroxamine mesylate might be in part due to inhibition of complex II activity. Taken together, our results suggest that complex II might be considered as one of the primary factors to regulate mitochondrial respiratory function by responding to the cellular iron level, thereby influencing cellular growth.