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First published online 10 June 2008
doi: 10.1242/jcs.026633

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PPAR accelerates cellular senescence by inducing p16^INK4 expression in human diploid fibroblasts

¹ Research Center on Aging, Department of Biochemistry and Molecular Biology, Peking University Health Science Center, 38 Xueyuan Road, Beijing 100083, People's Republic of China
² Cardiovascular Research Institute, Peking University Health Science Center, 38 Xueyuan Road, Beijing 100083, People's Republic of China

View larger version (48K): [in this window] [in a new window]   Fig. 1. Ligand-activated PPAR induced changes associated with senescence and induced irreversible cell-growth arrest in 2BS and WI-38 cells. 2BS and WI-38 cells transfected with the expression plasmids pcDNA3.1 (vector) or pcDNA-PPAR (PPAR) were analyzed for the relative senescence markers (all transformants of 2BS cells at PD42 and WI-38 cells at PD37). Cells were treated with 20 µM troglitazone or DMSO (vehicle) as indicated. (A) Western blot analysis of PPAR overexpression in PPAR-transfected cells compared with vector-transfected cells. Western blotting was performed using specific antibodies against PPAR as indicated. The β-actin lane serves as a loading control. (B) Vector-transfected and PPAR-transfected cells were stained for SA-β-gal activity (blue), a classical marker of senescence. (C) Growth curves of vector-transfected and PPAR-transfected cells were determined by the MTT assay. Values are the mean ± s.d. of triplicate points from a representative experiment (n=3), which was repeated three times with similar results. Values accompanied by different symbols are statistically significantly different from each other. (D) Flow-cytometry analysis of vector-transfected and PPAR-transfected cells. Each experiment was performed at least three times. The table shows the representative data. The graph depicts data from three independent experiments (means ± s.d.). *P0.05 vs vehicle-treated vector cells; #P0.001 vs vehicle-treated vector cells.

View larger version (53K): [in this window] [in a new window]   Fig. 2. The silencing of PPAR suppressed the senescence-associated features and cell proliferation in 2BS and WI-38 cells. 2BS and WI-38 cells transfected with the expression plasmids pSilencer 2.1-U6 neo (RNAi vector) or pSilencer-PPAR (siPPAR) were analyzed for the relative senescence markers (all transformants of 2BS at PD51 and WI-38 at PD47). Cells were treated with 20 µM troglitazone or DMSO (vehicle) as indicated. (A) Western blot analysis of PPAR silencing in siPPAR-transfected cells compared with RNAi-vector-transfected cells. Western blotting was performed using specific antibodies against PPAR as indicated. The β-actin lane serves as a loading control. (B) RNAi-vector-transfected and siPPAR-transfected cells were stained for SA-β-gal activity (blue), a classical marker of senescence. (C) Growth curves of RNAi-vector-transfected and siPPAR-transfected 2BS cells were determined by the MTT assay. Values are the mean ± s.d. of triplicate points from a representative experiment (n=3), which was repeated three times with similar results. Values accompanied by different symbols are statistically significantly different from each other. (D) Flow-cytometry analysis of RNAi-vector-transfected and siPPAR-transfected 2BS cells. Each experiment was performed at least three times. The table shows the representative data. The graph depicts data from three independent experiments (means ± s.d.). *P0.05 vs vehicle-treated RNAi-vector cells.

View larger version (19K): [in this window] [in a new window]   Fig. 3. PPAR activation increases p16 protein levels in 2BS and WI-38 cells. 2BS and WI-38 cells were transfected with the expression plasmids pcDNA3.1 (vector), pcDNA3.1-PPAR (PPAR), pSilencer 2.1-U6 neo (RNAi vector) or pSilencer-PPAR (siPPAR) and treated, as indicated, with 20 µM troglitazone or DMSO (vehicle) for 72 hours. (A) Western blot analysis of PPAR and p16INK4 expression in vector-transfected or PPAR-transfected cells. (B) Western blot analysis of PPAR and p16INK4 expression in RNAi-vector-transfected or siPPAR-transfected cells. Western blotting was performed using specific antibodies against PPAR and p16INK4 as indicated. The β-actin lane served as a loading control.

View larger version (38K): [in this window] [in a new window]   Fig. 4. PPAR binds to the PPRE-containing region in the p16 gene promoter. (A) Schematic diagram of the p16 gene promoter. `PPRE' denotes putative PPAR-binding sites and `control' denotes a region located immediately downstream of PPRE. The numbers are the positions upstream of the p16 gene translation initiation site. (B) Soluble chromatin was prepared from 2BS cells [young (Y; PD25) or senescent (S; PD62)] or WI-38 cells [young (Y; PD20) or senescent (S; PD50)] treated (+) or not (–) with 20 µM troglitazone for 48 hours. (C-E) Soluble chromatin was prepared from young or senescent 2BS cells treated or not with 20 µM troglitazone (C) or 10 µM pioglitazone (D,E) for 48 hours. Precipitated DNA samples were amplified with primers recognizing the PPRE domain. ChIP assays were quantified by real-time PCR (C,E). Values are expressed relative to the controls (untreated young 2BS cells), which were set as 1. Values are the mean ± s.d. of triplicate points from a representative experiment (n=3), which was repeated three times with similar results. *P0.05. (F) Soluble chromatin was prepared from young or senescent 2BS cells treated or not with 10 µM GW9662 for 48 hours. Precipitated DNA samples were amplified with primers recognizing the PPRE domain. (G) Soluble chromatin was prepared from young or senescent 2BS cells treated or not with 20 µM troglitazone for 48 hours. Precipitated DNA samples were amplified with primers recognizing the control element as indicated in A. IP was performed with an antibody against PPAR and DNA was amplified using primer pairs as indicated. As negative controls, the no antibody (No Ab) sample is an immunoprecipitation that did not contain antibody, and antibody against β-actin was used as an irrelevant antibody control. The input sample (Input) contained 0.5% of the total starting chromatin. The ChIP assays were repeated three times, and results of a representative experiment are shown.

View larger version (10K): [in this window] [in a new window]   Fig. 5. Induction of human p16 promoter activity by ligand-activated PPAR. (A) 2BS and WI-38 cells were transfected with expression plasmids pcDNA3.1 (vector) or pcDNA-PPAR (PPAR) as indicated, together with wild-type p16-Luc (p16 WT) or mutant p16-Luc (p16 mutant). (B) 2BS cells were transfected with expression plasmids pSilencer 2.1-U6 neo (RNAi vector) or pSilencer-PPAR (siPPAR) as indicated, together with wild-type p16-Luc (p16 WT). Cells were subsequently treated with 20 µM troglitazone, 10 µM pioglitazone or 10 µM GW9662. Luciferase activities were then measured 24 hours after treatment. Values are the mean ± s.d. of triplicate points from a representative experiment (n=3), which was repeated three times with similar results. *P0.05.

View larger version (22K): [in this window] [in a new window]   Fig. 6. The silencing of p16 in 2BS cells prevented the appearance of senescence-associated features and the growth-inhibition effects of PPAR agonists. 2BS cells transfected with the expression plasmids pSilencer 2.1-U6 neo (RNAi vector) or pSilencer-p16 (sip16) were analyzed for the relative senescence markers (all at PD51). Cells were treated with 20 µM troglitazone or DMSO (vehicle) as indicated. (A) Western blot analysis of p16 silencing in sip16-transfected cells compared with RNAi-vector-transfected cells. Western blotting was performed using specific antibodies against p16INK4 as indicated. The β-actin lane serves as a loading control. (B) RNAi-vector-transfected and sip16-transfected cells were stained for SA-β-gal activity (blue), a classical marker of senescence. (C) Growth curves of RNAi-vector-transfected and sip16-transfected 2BS cells were determined by the MTT assay. Values are the mean ± s.d. of triplicate points from a representative experiment (n=3), which was repeated three times with similar results. Values accompanied by different symbols are statistically significantly different from each other. (D) Flow-cytometry analysis of RNAi-vector-transfected and sip16-transfected 2BS cells. Each experiment was performed at least three times. The table shows the representative data. The graph depicts data from three independent experiments (means ± s.d.). *P0.05 vs vehicle-treated RNAi-vector cells.

View larger version (26K): [in this window] [in a new window]   Fig. 7. The expression level of PPAR. (A) The relative amounts of p16, PPAR and GAPDH mRNA in young (Y; PD25), middle-aged (M; PD42) and senescent (S; PD62) 2BS cells. Total RNA was isolated as indicated and then subjected to RT-PCR analysis using PPAR or p16 primers. GAPDH was used as an internal control for normalization purposes. (B) Western blot analysis of PPAR and p16INK4 expression in 2BS and WI-38 cells. Total proteins were extracted, and western blotting was performed using specific antibodies against PPAR and p16INK4 as indicated. The β-actin lane serves as a loading control.

View larger version (24K): [in this window] [in a new window]   Fig. 8. Phosphorylation represses the transactivating function of PPAR. (A) Western blot analysis of phosphorylation levels of PPAR in young and senescent 2BS and WI-38 cells. Total proteins were extracted, and western blotting was performed using specific antibodies against PPAR and phosphorylated PPAR (p-PPAR) as indicated. Arrow indicates phosphorylated PPAR, and the observed doublet is reported as two translation initiation sites. (B) 2BS cells were transfected with expression plasmids pcDNA3.1 (–), pcDNA-PPAR (WT) or mutant pcDNA-PPAR (S84A) as indicated, together with wild-type p16-Luc. Cells were subsequently treated with 20 µM troglitazone or 10 µM pioglitazone. Luciferase activities were measured 24 hours after treatment. Values are the mean ± s.d. of triplicate points from a representative experiment (n=3), which was repeated three times with similar results. *P0.05.

View larger version (12K): [in this window] [in a new window]   Fig. 9. Model of PPAR-regulated cellular senescence.

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