Crosstalk between age accumulated DNA-damage and the SIRT1-AKT-GSK3ß axis in urine derived renal progenitor cells

UdRPCs from aged donors show typical hallmarks of aging

We recently reported MSCs isolated directly from urine samples. These cells express the renal stem cell markers SIX2, CITED1 WT1, CD133, CD24 and CD106, we referred to these cells as urine derived renal progenitor cells (UdRPCs) [34]. In this study, the progenitor cells were isolated from distinct individuals of mixed ethnicity with ages ranging from 21 to 77 years. It is well documented in the literature, that MSCs show decline of self-renewal capacity and of immunosuppressive properties with increased donor age and in vitro expansion [35–38]. As previously reported UdRPCs can be kept in culture for up to 12 passages, whereas cells from aged donors show a decline of proliferation capacity after 9-10 passages. Therefore, all experiments were carried out with UdRPCs after 7-8 passages. Hierarchical clustering analysis comparing the transcriptomes of UdRPCs revealed a distinct expression pattern of cells derived from donors aged between 21 to 51 years (young, green box) and 54 to 61 years (aged, red box) (Figure 1A). Microarray analysis revealed a common expressed gene-set of 11917 between UdRPCs derived from young- and elderly donors, while 750 genes were exclusively expressed in cells derived from young- and 155 genes in cells derived from elderly individuals by comparing the expressed gene set (det-p < 0.05) (Figure 1B). The most over-represented GO BP-terms common expressed in UdRPCs derived from young- and elderly donors are associated with metabolic processes such as, organic acid transport and regulation of ion transport as well as cell junction organization and cell morphogenesis involved in differentiation. The most over-represented GO BP-terms exclusive to the young UdRPCs are associated with DNA-replication, mesodermal cell differentiation, renal system development and PI3K-AKT signalling pathway. In comparison the most over-represented GO BP-terms exclusive to aged UdRPCs are associated with assembly of collagen fibrils and other multimeric structures and regulation of calcium ion transport (Figure 1C). The full gene list can be found in Supplementary Table 3. Additionally, we analysed our data-set for genes associated with the hallmarks of aging as proposed by Otin et al., (Figure 1D) [4]. The heatmap reveals a negative correlation between the donor age and the expression of genes involved in genomic instability (ATM, MSH6, TFEB and XRCC1), epigenetic alterations (SETDB1, KDM6A, EZH1, SIRT1, SETDB2, HDAC6, HDAC4, KDM4B and SIRT3) and genes involved in de-regulation of nutrient sensing of the one carbon-, cysteine- and methionine-metabolic pathways (SHMT1, SHMT2 and MAT2B). Furthermore, our transcriptome data reveals a positive correlation between donor age and the expression of genes involved in cellular senescence (CDKN1A, CDKN2A and CDKN2D) and stem cell exhaustion (CXCL1, IL6 and IL8). In conclusion, the transcriptome data revealed typical hallmarks of aging in UdRPCs derived from elderly donors.

Figure 1. Comparative transcriptome and gene ontology analysis of urine-derived renal progenitors from young and aged donors. A hierarchical cluster dendrogram revealed distinct clusters of UdRPCs derived from young and aged donors (A). Expressed genes (det-p < 0.05) in UdRPCs and podocytes compared in the Venn diagram (B), shows distinct (750 in young and 155 in aged UdRPCs) and overlapping (11917) gene expression patterns. The most over- represented GO BP-terms exclusive in either young or old UdRPCs are shown in (C) and including DNA replication and renal development for the young UdRPCs and assembly of collagen fibrils and other multimeric structures and regulation of calcium ion transport for the old UdRPCs. The heatmap (D) reveals a distinct expression pattern between young and old UdRPCs for genes encoding for the following hallmarks of aging: genomic instability, epigenetic alterations, deregulation of nutrient sensing, cellular senescence, and stem cell exhaustion.

UdRPCs show decline of stem cell characteristics and an increase of DNA-damage with increase donor age

Since our microarray data revealed genes encoding for mesodermal cell differentiation and renal system development to be exclusively expressed in UdRPCs derived from young donors, we assumed that this is caused by an age-associated decline of self-renewal capacity. To test this hypothesis, we applied immunofluorescent staining for the renal stem cell marker SIX2 and qRT-PCR analysis for the stem cell markers SIX2 and CD133. Surprisingly, the immunofluorescent staining revealed that almost 100% of the isolated cells from donors aged between 21 and 51 years are positive for SIX2 (Figure 2B). In contrast, qRT-PCR analysis revealed a significant downregulation/ 0.98-fold (p < 0.01) of SIX2 mRNA expression between cells derived from the 21-year-old donor compared to cells derived from all other donors. For the stem cell marker CD133 qRT-PCR analysis revealed a significant downregulation/ 1.16-fold (p ≤ 0.01) of mRNA expression between cells derived from donors aged between 21 and 48 compared to individuals aged between 51 and 77 years (Figure 2B). As a further marker of premature terminal differentiation and/or senescence [39] we assessed the truncated form of the Lamin A transcript Progerin by semi-quantitative PCR. Strikingly, we found an increase of truncated Lamin A transcript Progerin within UdRPCs derived donors aged between 21 and 48 compared to individuals aged between 51 and 77 years) (Figure 2C). Accumulation of Progerin has been described to lead to DNA-damage and chromosomal aberrations [40, 41], by inhibiting inter alia the SIRT6 mediated DNA-damage repair mechanism [42]. To test our hypothesis that the identified increase of Progerin mRNA in the aged UdRPCs is accompanied by increased DNA-damage, we analysed phosphorylation levels of Histone 2A (pH2A.X), an established biomarker of DNA-damage at DBS [43], by immunofluorescent based detection (Figure 2D). UdRPCs derived from individuals aged between 21 and 45 years showed a positive pH2A.X staining only in a small percentage of cells, 2% and 6% respectively. In contrast we detected a significant (p = 0.03) increase of DBS in cells derived from donors aged 51, 63 and 77 years, with 28%, 50% and 55% of cells being positive for the pH2A.X staining (Figure 2D and Supplementary Figure 1A). Furthermore, we applied qRT-PCR analysis for ATM, P16 and TP53 (Figure 2E and Supplementary Figure 1B). mRNA expression of CDKN2A and TP53 were found to be not significantly altered between UdRPCs derived the different donors (p-value P16: p = 0.91 and TP53: p = 0.41), with a trend for P16 being up- and TP53 being downregulated with increased donor age but could also reflect heterogeneity in-between individuals. In contrast, qRT-PCR analysis revealed a significant downregulation/ 0.85-fold (p < 0.05) of ATM mRNA expression between cells derived from the 21-years-old donor compared to UdRPCs derived from individuals aged between 27 and 77 years. Finally, we evaluated the expression of Methionine Adenosyltransferase 2B (MAT2b) by qRT-PCR. This enzyme catalyzes the final step of one carbon metabolism by forming S-Adenosyl-L-methionine from methionine and adenosine triphosphate. For MAT2b a significant downregulation/ 1.15-fold of mRNA levels were found in the UdRPCS derived from donors aged between 21 and 63 compared to donors aged 69 to 77 (p < 0.01) (Supplementary Figure 1B). In summary, UdRPCs show an increase in DNA damage with increased donor age, while MSC markers CD133 and the renal progenitor marker SIX2 were found to be downregulated.

Figure 2. UdRPCs show decline of mesenchymal stem cell characteristics and an increase of DNA-damage with increase donor age. UdRPCs from donors aged between 21 and 77 years were isolated and stemness status was confirmed by immunofluorescent staining for the renal progenitor marker SIX2 (A) (scale bars: 100 μm). mRNA expression of SIX2 and the stem cell proliferation marker CD133 was determined by quantitative real time PCR (B). RT-PCR analysis reveal Progerin transcripts in UdRPCs (C). UdRPCs from donors aged between 21 and 77 years were isolated and DNA damage was visualized by immunofluorescence-based staining for phosphorylated Histone2A.X (pH2A.X) (D). mRNA expression of ATM was determined by quantitative real time PCR (E).

Age associated changes in the SIRT1-AKT-GSK3ß regulatory axis

SIRT1 [30] and the AKT pathway [14] are involved in DNA-damage-repair and our results indicate a downregulation of SIRT1 and members of the AKT pathway in UdRPCs derived from individuals aged 48 years and above. Therefore, we evaluated the expression of SIRT1 as well as the phosphorylation levels of H2A.X, AKT and its downstream target GSK3ß by Western blot detection (Figure 3A). Consistently, we found SIRT1 expression exclusively in the UdRPCs derived from individuals aged ≤ 48 years. Normalized to ß-actin expression we found a significant reduction of SIRT1 protein expression (p = 0.01) in UdRPCs derived from elderly donors by 81.3% and 64.7% (48 and 51 years). Our Western blot results revealed a similar observation for the phosphorylation levels of AKT and GSK3β, while total levels of AKT and GSK3ß expression were found to be unchanged. The ratio of the phosphorylated version to the total protein levels revealed a significant reduction of AKT (p = 0.05) and GSK3β (p = 0.04) protein phosphorylation in UdRPCs derived from donors aged 48 and 51 years, by 97.36% and 97.62% for AKT and 55.69% und 55.95% for GSK3β. Furthermore, we also evaluated H2A.X phosphorylation and observed an increase of DBS only in the sample derived from the 51-year-old individual. Normalized to ß-actin the increase was found to be not significant (p = 0.47) but became highly significant when H2A.X phosphorylation was normalized to the detected SIRT1 protein expression (p ≤ 0.01). To further confirm that SIRT1 becomes downregulated in UdRPCs derived from aged donors, we applied qRT-PCR analysis. According to the genecards database the SIRT1 mRNA has six major splicing variants. Almost all splicing variants consist of exon 4 to exon 6 and can be distinguished into two groups by the existence of exon 1-3 or exon 7-10 (Supplementary Figure 2). To evaluate which is the major splicing variant that changes in UdRPCs obtained from elderly donors, we designed primers that anneal in exon 1 and 2 as well as exon 7 and 8 (Supplementary Table 1). Surprisingly, we found both variants to be expressed and significantly altered (Exon 1-2: p = 0.03 and Exon 7-8: p = 0.01) between cells derived from the 21- and the 27-year-old donor compared to the UdRPCs derived from donors aged between 45 and 77 years, with fold changes of 0.54 and 0.75 respectively (Figure 3B). It has been recognized, that during the aging process “de novo” DNA methylation occurs within the promoter region of transcriptional downregulated genes [44, 45]. By applying genomic bisulfite sequencing we analysed the methylation status of the SIRT1 gene in UdRPCs derived from the 27-year- and the 51-year-old individual. In total a 341bp fragment of the SIRT1 promoter containing 45 CpG-dinucleotides were analysed. Stinkingly, we found 15.2% of CpGs to be methylated in the UdRPCs derived from the 27-year-old individual, while UdRPCs derived from the 51-year-old individual showed 61.3% of CpGs to be methylated. We concluded that UdRPCs could be distinguished in high and low SIRT1 expressing cells, depending on the donor age with a threshold of 48 years from our cohort. Finally, we wanted to investigate whether the age associated downregulation of SIX2, which is needed to maintain renal progenitor cells during kidney organogenesis [34], affects the level of SIRT1 mRNA expression rate. Since the UdRPCs derived from the 27-year-old individual showed the highest SIRT1 protein expression in Western blot analysis, we choose this cell line for Immunoprecipitation analysis. SIX2 has been reported to regulate the expression of Odd-skipped related 1 (Osr1) [46] and Glial Cell Derived Neurotrophic Factor (GDNF) [47] so we chose these as positive controls, while RPL0 was chosen as negative control. PCR analysis was performed with DNA derived from the whole cell extract (Input), after Immunoprecipitation with (IP) and without (negative control) antibodies against SIX2. Hereby, we could confirm a direct interaction between the SIX2 transcription factor and the genomic DNA of GDNF, OSR1 and SIRT1 (Figure 3D). We hypothesize that for the aging process of UdRPCs, the renal progenitor transcription factor SIX2 and SIRT1 mutually influence their transcriptional levels positively in cells derived from donors aged ≤ 48 years. In cells aged >48 and above we found the transcriptional downregulation of SIX2 and SIRT1, for which DNA-Methylation seems causative in the latter case. This downregulation of SIRT1 is associated with reduced phosphorylation levels of AKT and GSK3ß and renders the cells more vulnerable to double strand breaks.

Figure 3. SIX2/SIRT1/AKT/GSK3β network is altered in UdRPCs derived from aged donors. Relative protein expression normalized to ß-ACTIN for SIRT1, AKT and GSK3ß and relative protein phosphorylation for AKT, GSK3β and pH2A.X was detected by Western blot (A). mRNA expression of SIRT1 was determined by quantitative real time PCR (B). Detailed analyses by bisulfite sequencing of CpG island methylation patterns within 5′ regulatory region of SIRT1 gene in young and aged UdRPCs (C). SIX2 binding within the SIRT1 gene was confirmed by Immunoprecipitation followed by PCR analysis (D). The grey dashed line indicates that gel picture has been merged (for the original gel picture see Supplementary Figure 6).

DNA damage dependent activation of the SIX2/SIRT1/AKT/GSK3ß network by resveratrol

Resveratrol has been recognized as a potent activator of SIRT1 [48, 49]. Therefore, we evaluated by qRT-PCR the effect of varying concentrations of resveratrol on low SIRT1 expressing UdRPCs with and without age-associated DNA damage. We prepared final concentrations of resveratrol directly in the cell culture medium ranging from 0.1μM to 250μM and treated the UdRPCs derived from the 48- and 51-year- old individual for 24h. Strikingly we detected a significant increase of SIRT1 Exon 7-8 mRNA (48: p = 0.05; 9-fold and 51: p = 0.04; 0.44-fold) and SIRT1 Exon 1-2 only in the UdRPCs derived from the 51-year-old-individual (p < 0.01; 0.4-fold) (Figure 4B, 4F and Supplementary Figure 3A). In contrast, the resveratrol treatment caused a significant downregulation of SIRT1 Exon 1-2 mRNA in the UdRPCs derived from the 48-year-old-individual (p = 0.02; 0.52-fold) (Supplementary Figure 3A). Immunofluorescence-based detection and Western blot analysis revealed a significant upregulation of SIRT1 protein by 50% and 120% respectively (48: p = 0.04 and 51: p = 0.03) for both cell lines treated with 1μM resveratrol (Figure 3A, 3D, 3H and Supplementary Figure 3A). In contrast a significant downregulation of SIRT1 mRNA (48: p < 0.01 and 51: p < 0.01) and protein (48: p < 0.01) was detected within the cells treated with the resveratrol solutions containing 2.5μM and higher concentrations by 100 and 50% respectively (Figure 4D, 4H and Supplementary Figure 3A). To test if this effect is due to the resveratrol, we also measured the expression level of MAT2B. The promoter region of this gene has been identified to harbors two resveratrol binding pockets and gets activated by resveratrol in a time- and dose-dependent manner [50]. As expected MAT2B mRNA expression levels were significantly upregulated (48: p < 0.01 / 1.2-fold change and 51: p < 0.01 / 1.24-fold change) in the UdRPCs treated with low- concentrations and significantly downregulated (48: p < 0.01 / 0.97-fold change and 51: p < 0.01 / 0.27-fold change) in the cells treated with the high concentrations of resveratrol. These results correlate with the observed changes in SIRT1 mRNA expression (Supplementary Figure 3B). Furthermore, immunofluorescence- based and Western blot detection of pH2A.X revealed no DBS in UdRPCs derived from the 48-year-old individual under control conditions and in the cells treated with the 1μM resveratrol solution, but a significant increase of DBS (p < 0.01; 86% of cells were found to be pH2A.X positive) was detected when cells were treated with the 2.5μM resveratrol solution (Figure 4A, 4D). Accordingly, 54% of UdRPCs obtained from the 51-year-old individual revealed a positive pH2A.X staining under control conditions, which became significantly elevated (51: p = 0.05; 71% of cells were found to be pH2A.X positive) by the 2.5μM resveratrol solution (Figure 4A, 4H). Strikingly only 34% of cells treated with 1μM resveratrol were positive for pH2A.X expression (51: p = 0.05). To test our hypothesis if resveratrol induced activation of SIRT1 prevents cellular senescence by increasing DNA damage repair mechanisms, we evaluated mRNA expression levels of ATM and the cell cycle regulator P16 by qRT-PCR (Figure 4B, 4F and Supplementary Figure 3B). We found a significant upregulation / 26-fold of mRNA expression in the UdRPCs derived from the 48-year-old donor treated with low concentrations and a significant downregulation/ 0.99-fold and 0.5-fold in cells derived from both donors 48 and 51 treated with high concentrations of resveratrol (48: p < 0.01, 51: p < 0.01). In contrast P16 expression levels became significantly downregulated/ 0.62-fold in UdRPCs derived from the 48-year-old donor with the low concentration and significantly upregulated 8.8-fold when cells were treated with the high concentrations of resveratrol (48: p < 0.01). Next, we assessed the effects of resveratrol on the stem cell characteristics by measuring the expression level of the renal progenitor marker SIX2 and CD133 (Figure 4A, 4F and Supplementary Figure 3B) as well as the occurrence of the aberrant Lamin A splicing variant Progerin (Figure 4E, 4I). In both cell cultures we found a non-significant upregulation/ 0.35-fold and 0.16-fold of SIX2 mRNA (48: p < 0.31 and 51: p < 0.27) when cells were treated with the low concentrations of resveratrol, while high concentrations of resveratrol significantly downregulated/ 0.99-fold and 0.45-fold (48: p < 0.01 and 51: p < 0.01) SIX2 mRNA expression levels. In contrast for CD133 a significant upregulation/ 5.3-fold was only found in the UdRPCs derived from the 48-year-old individual (p = 0.02) when treated with the low and a significant downregulation/ 0.83-fold (p < 0.01) when treated with the high concentrations of resveratrol. UdRPCs derived from the 51-year-old individual showed no difference in CD133 mRNA expression when treated with the low and a slightly but not significant (p = 0.2) downregulation/ 0.31-fold of CD133 when treated with the high concentration of resveratrol. The aberrant Lamin A splicing variant Progerin could not be detected in the UdRPCs derived from the 48-year-old individual treated with 0μM, 0.1μM or 1μM of resveratrol, but alternate splicing of Lamin A occurred when cells were treated with the 2.5μM resveratrol solution. In contrast, in the UdRPCs derived from the 51-year-old individual the two aberrant splicing variants were detected in all samples, but in cells treated with low concentrations of resveratrol the intensity of one of the detected bands became much weaker. Next, we performed Western blot analysis of the downstream targets of SIRT1, namely AKT and GSK3ß (Figure 4D, 4H and Supplementary Figure 3A). For both cell lines we did not observe a change in total AKT nor GSK3ß protein expression in either treatment. We found that in the low DNA damage UdRPCs derived from the 48-year-old individual GSK3β-phosphorylation (p = 0.04) was significantly increased by 32% when the cells were treated with the 1μM solution of resveratrol. Furthermore, within cells from the 48-year-old individual treated with the 2.5μM solution of resveratrol GSK3β phosphorylation was found to be significantly decreased by 56% (p < 0.01). In contrast, AKT phosphorylation was found to be significantly decreased when cells were treated with either of the resveratrol solutions by 41.19% and 67.86% respectively (p < 0.01). In the high DNA damage UdRPCs derived from the 51-year-old individual no significant changes in AKT or GSK3ß phosphorylation were observed. Finally, we evaluated the effects of the resveratrol treatment on the proliferative capacity of UdRPCs (Figure 4C, 4G). After 24h cells from both donors treated with the 2.5 μM solution of resveratrol showed a significant decrease in cell number (p < 0.01) (Figure 4G). In contrast only the UdRPCs derived from the 48-year-old individual showed a significant increase in cell number after 24h of 0.1 and 1μM resveratrol treatment (Figure 4C). In summary, resveratrol can activate SIRT1 in a dose dependent manner in UdRPCs. A high dose >2.5μM enhances age- associated phenotypes whereas low doses <1μM induce the opposite effect.

Figure 4. The SIX2/SIRT1/AKT/GSK3β network can be activated by resveratrol and regulates the cell fate of UdRPCs. Cell culture medium of UdRPCs was supplemented with different concentrations of resveratrol for 24h. Activation of SIRT1 and phosphorylation of H2A.X were monitored by immunofluorescence-based detection (A) (scale bars: 100 μm). mRNA expression of SIRT1, ATM, P16 and SIX2 was determined in U48 by quantitative real-time PCR (B). Cell growth of U48 was evaluated after 24h of resveratrol treatment as depicted (C). Relative protein expression normalized to ß-ACTIN for SIRT1, AKT and GSK3ß and relative protein phosphorylation for AKT, GSK3ß and pH2A.X in U48 was detected by Western blot (D). RT-PCR analysis reveal Progerin transcripts in U48 treated with high concentrations of resveratrol (E). mRNA expression of SIRT1, ATM, P16 and SIX2 was determined in U51 by quantitative real-time PCR (F). Cell growth of U51 was evaluated after 24h of resveratrol treatment as depicted (G). Relative protein expression normalized to ß-ACTIN for SIRT1, AKT and GSK3ß and relative protein phosphorylation for AKT, GSK3ß and pH2A.X in U51 was detected by Western blot (H). RT-PCR analysis reveal Progerin transcripts in U51 treated with high concentrations of resveratrol (I).

DNA damage induces an aging phenotype by downregulation of SIRT1

DNA damage can trigger four potential outcomes namely, transient cell cycle arrest coupled with DNA repair, apoptosis, senescence or cell differentiation [9]. SIRT1 has been reported to participate in all of the mentioned biological processes, so we tested the effect of endogenous induced DNA damage on SIRT1 expression in UdRPCs, expressing high levels of SIRT1 [20–22]. Bleomycin has been recognized as potent inducer of DBS for many years [51, 52], so we prepared final concentrations of 1μM resveratrol, 30μg/ml Bleomycin and a combination of both substances directly in the cell culture medium and treated the UdRPCs derived from the 27-year- old individual for 24h. Interestingly while resveratrol treated UdRPCs showed a significant increase/ 0.41-fold in SIRT1 mRNA expression (p < 0.01), upon resveratrol treatment (Figure 5B), we found SIRT1 protein expression to be unchanged between resveratrol treated and control cells (Figure 5C and Supplementary Figure 4B). In contrast, cells treated with the combination of resveratrol and bleomycin showed no changes in mRNA expression, while bleomycin alone showed a significant decrease/ 0.21-fold in SIRT1 mRNA as well as protein expression (p < 0.01) (Figure 5C and Supplementary Figure 4B). Immunofluorescence- based detection of H2A.X phosphorylation revealed no beneficial effect of the resveratrol treatment alone, with 34% (control) and 33% (1μM resveratrol) of cells being positive for DBS. In contrast UdRPCs treated with either Bleomycin alone or the combination of 1μM resveratrol and 30μg/ml Bleomycin showed a significant increase (p < 0.01), with 78% and 59% of cells being positive for pH2A.X expression (Figure 5A). Furthermore, Western blot analysis of H2A.X phosphorylation normalized either to ß-actin or SIRT1 expression revealed a significant increase (p < 0.01) of DBS in cells treated with Bleomycin (Figure 5C). In contrast H2A.X phosphorylation could not be detected in either the control, the resveratrol, and the resveratrol + Bleomycin treated samples (Figure 5C). Strikingly the increased H2A.X phosphorylation was accompanied by a significant increase/ 9.23-fold of P16 mRNA expression, while for all other conditions expression levels were found to be unchanged (p < 0.01). Surprisingly ATM mRNA expression levels were also found to be unchanged, with a slight downregulation in the resveratrol treated samples. Next, we assessed the effects of the resveratrol and/or Bleomycin on the stem cell characteristics of UdRPCs by measuring the expression level of the renal progenitor marker SIX2 and CD133 (Figure 5B). In accordance with our previous data, UdRPCs treated with resveratrol alone and in combination with Bleomycin showed an upregulation of SIX2 (1.38-fold) and CD133 (4-fold) mRNA levels. While this upregulation was found to be not significant when cells were treated with the combination of Resveratrol and Bleomycin (p = 0.25), the upregulation became highly significant by resveratrol treatment alone (p = 0.01). In contrast bleomycin treatment alone slightly downregulated mRNA expression levels of SIX2 and CD133 (Figure 5B and Supplementary Figure 4A). Finally, we performed western blot analysis of the downstream targets of SIRT1, namely AKT and GSK3ß (Figure 5C and Supplementary Figure 4B). Total levels of AKT were found to be significantly downregulated by 53.14% in the bleomycin treated cells, while total levels of GSK3ß were found to be significantly upregulated by 114% in the resveratrol treated cells. While AKT phosphorylation was found to be significantly upregulated in resveratrol only treated cells by 108% (p=0.05). UdRPCs treated with the combination of resveratrol and bleomycin and the bleomycin alone treated cells did not show a significant change. Interestingly GSK3β phosphorylation was found to be significantly downregulated in UdRPCs derived from the 27-year-old individual under all culture conditions (by 71.78% with Resveratrol, by 77.8% with Resveratrol and Bleomycin and 94.36% with Bleomycin) (Figure 5B and Supplementary Figure 4A). In conclusion, DNA-damage can induce an aging phenotype by downregulation of SIRT1.

Figure 5. DNA damage induces an aging phenotype by downregulation of SIRT1. Cell culture medium of UdRPCs was supplemented with 1μM of resveratrol and or 30 μg/ml of bleomycin for 24h. Phosphorylation of H2A.X was monitored by immunofluorescence-based expression analysis (A) (scale bars: 100 μm). mRNA expression of SIRT1, ATM, P16 and SIX2 was determined by quantitative real time PCR (B). Relative protein expression normalized to ß-ACTIN for SIRT1, AKT and GSK3ß and relative protein phosphorylation for AKT, GSK3β and pH2A.X was detected by Western blotting (C).

Cell culture conditions

UdRPCs were isolated as described in Rahman et al. [34] and were cultured in Proliferation Medium (PM) composed of 50% DMEM high Glucose and 50% Keratinocyte medium supplemented with 5% FCS, 0.5% NEAA, 0.25% Gtx and 0.5% Penicillin and Streptomycin at 37° C (Gibco, Carlsbad, CA, USA) under hypoxic conditions. For all experiments cells were collected after 7-8 passages and seeded in 6- or 12-well plates coated with 0.2% Gelatin (Thermo Fisher Scientific, Waltham, MA, USA). Resveratrol (Sigma Aldrich, St. Louis, MO, USA) and Bleomycin (Sigma Aldrich) were added to the the culture medium to a final concentration of 30 μg/ml. Cells were incubated with Resveratrol and Bleomycin containing culture medium for 24h.

Relative quantification of aging-associated gene expression by real-time PCR

Total RNA was extracted from UdRPCs using the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. First-strand cDNA synthesis was performed from 1 μg RNA by reverse transcription using oligo(dT) (Promega, Madison, WI, USA) and Moloney murine leukemia virus reverse transcriptase (Promega) in a volume of 50 μL at 42° C for 1 h.

Real time PCR of aging associated gene expression was performed as follows:

Real time measurements were carried out on the Step One Plus Real Time PCR Systems using MicroAmp Fast optical 384 Well Reaction Plate and Power Sybr Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA). The amplification conditions were denaturation at 95° C for 13 min. followed by 37 cycles of 95° C for 50s, 60° C for 45s and 72° C for 30s. Primer sequences are listed in Supplementary Table 1.

Immunofluorescence staining

Cells were fixed with 4% paraformaldehyde (PFA) (Polysciences, Warrington, PA, USA). Unspecific binding sites of the fixed cells were blocked by incubation with blocking buffer containing 10% normal goat or donkey serum, 1% BSA, 0.5% Triton, and 0.05% Tween, for 2h at room temperature. The primary antibody was diluted 1:1 in blocking buffer with PBS and incubated at 4° C overnight (or at least 16h). After incubation the cells were washed three times with PBS/0.05% Tween and the secondary antibodies were diluted the same way as the primary antibodies with a 1:500 dilution. After 1h of secondary antibody incubation the cells were washed again three times with PBS/0.05% Tween and nuclei were stained with Hoechst 1:5000 (Thermo Fisher Scientific) and cytoskeleton was stained with Alexa Flour 488 phalloidin (Thermo Fisher Scientific) (1:400). Images were captured using a fluorescence microscope (LSM700; Zeiss, Oberkochen, Germany) with Zenblue software (Zeiss). Individual channel images were processed with Fiji. Detailed Information of the used antibodies are given in Supplementary Table 2.

Microarray data analyses

Total RNA (1 μg) preparations were hybridized on the PrimeView Human Gene Expression Array (Affymetrix, Thermo Fisher Scientific, USA) at the core facility Biomedizinisches Forschungszentrum (BMFZ) of the Heinrich Heine University Düsseldorf. The raw data was imported into the R/Bioconductor environment and further processed with the package affy using background-correction, logarithmic (base 2) transformation and normalization with the Robust Multi-array Average (RMA) method. The heatmap.2 function from the gplots package was applied for cluster analysis and to generate heatmaps using Pearson correlation as similarity measure. Gene expression was detected using a detection-p-value threshold of 0.05. Differential gene expression was determined via the p-value from the limma package which was adjusted for false discovery rate using the q value package. Thresholds of 1.33 and 0.75 were used for up-/down-regulation of ratios and 0.05 for p-values. Venn diagrams were generated with the Venn Diagram package. Subsets from the venn diagrams were used for follow-up GO and pathway analyses as described by Zhou et al. [74]. Gene expression data will be available online at the National Centre of Biotechnology Information (NCBI) Gene Expression Omnibus.

Western blot analysis

UdRPCs were lysed in lysis buffer composed of 5M NaCl, 1% NP-40, 0.5% DOC, 0.1% SDS, 1 mM EDTA, 50mM Tris, pH 8.0, and freshly added 10μL/mL protease- and phosphatase inhibitor (Sigma Aldrich). 20μg of the obtained protein lysate was resolved in a 10% sodium dodecyl sulfate-PAGE gel and transferred onto Immobilon-P membrane (Merck Millipore, Burlington, MA, USA). Membranes were probed with primary antibody at 4° C overnight, washed three times with 0.1% Tween-20 in Tris-buffered saline, and incubated with secondary antibody for 1h at room temperature. The signals were visualized with enhanced luminescence Western Bright Quantum (Advansta, San Jose, CA, USA). Detailed Information of the used antibodies are given in Supplementary Table 2.

Expression of Lamin A and Progerin

Total RNA was extracted from UdRPCs using the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. First-strand cDNA synthesis was performed from 1μg RNA by reverse transcription using oligo(dT) (Promega, Madison, WI, USA) and Moloney murine leukemia virus reverse transcriptase (Promega) in a volume of 50μL at 42° C for 1 h. Lamin A and Progerin were detected as described by McClintock et al., [75]. Primer sequences are listed in Supplementary Table 1.

Immunoprecipitation

Cells were chemically crosslinked with 11% formaldehyde solution for 15min at room temperature. Cells were washed twice with 1× PBS and harvested using a silicon scraper in a lysis buffer, and genomic DNA was sonicated at 4° C in TPX® polymethylpentene tubes using a Bioruptor® sonicator (Diagenode, Liege, Belgium). Twenty sonication pulses of each 15sec were applied. The resulting wholecell extract (WCE) was incubated overnight at 4° C with 100μl of Dynal Protein A magnetic beads (Diagenode) previously pre-incubated with (input) and without (negative control) 10μg of SIX2 antibody. Beads were washed five times with RIPA buffer and once with TE containing 50mM NaCl. Bound complexes were eluted from the beads by heating at 65° C with occasional vortexing, and crosslinking was reversed by overnight incubation at 65° C. Input and negative control were also treated for crosslink reversal. Immunoprecipitated DNA and whole-cell extract DNA were then purified by treatment with RNase A, proteinase K, multiple phenol: chloroform:isoamyl alcohol extractions and precipitation with ethanol. Purified DNA was amplified using the PCR protocol.

Bisulfite sequencing

Bisulfite sequencing was performed following bisulfite conversion with the EpiTec Kit (Qiagen, Hilden, Germany) as described in Erichsen et al. [70]. PCR primer sequences are given in Supplementary Table 1 and refer to +1 transcription start of the following sequences:

Homo sapiens sirtuin 1 (SIRT1), RefSeqGene on chromosome 10

NCBI Reference Sequence: NG_050664.1

Obtained sequences were analysed using Quma (http://quma.cdb.riken.jp/) as described in [76].

Statistical analysis

Data is presented as arithmetic means + standard error of mean. At least three experiments were used for the calculation of mean values. To address the statistical significance, we applied the two-samples Student’s t-test with a significance threshold 0.05. The level of significance was set to p < 0.05.