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

Journal of Cell Science 121, 2235-2245 (2008)
Published by The Company of Biologists 2008
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Research Article

PPAR{gamma} accelerates cellular senescence by inducing p16INK4{alpha} expression in human diploid fibroblasts

Qini Gan1, Jing Huang1, Rui Zhou1, Jing Niu1, Xiaojun Zhu2, Jing Wang1, Zongyu Zhang1 and Tanjun Tong1,*

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

* Author for correspondence (e-mail: ttj{at}bjmu.edu.cn)

Accepted 14 April 2008


   Summary
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 Summary
Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) plays an important role in the inhibition of cell growth by promoting cell-cycle arrest, and PPAR{gamma} activation induces the expression of p16INK4{alpha} (CDKN2A), an important cell-cycle inhibitor that can induce senescence. However, the role of PPAR{gamma} in cellular senescence is unknown. Here, we show that PPAR{gamma} promotes cellular senescence by inducing p16INK4{alpha} expression. We found several indications that PPAR{gamma} accelerates cellular senescence, including enhanced senescence-associated (SA)-β-galactosidase staining, increased G1 arrest and delayed cell growth in human fibroblasts. Western blotting studies demonstrated that PPAR{gamma} activation can upregulate the expression of p16INK4{alpha}. PPAR{gamma} can bind to the p16 promoter and induce its transcription, and, after treatment with a selective PPAR{gamma} agonist, we observed more-robust expression of p16INK4{alpha} in senescent cells than in young cells. In addition, our data indicate that phosphorylation of PPAR{gamma} decreased with increased cell passage. Our results provide a possible molecular mechanism underlying the regulation of cellular senescence.

Key words: PPAR{gamma}, p16INK4{alpha}, Cellular senescence, Fibroblast


   Introduction
Top
 Summary
 Introduction
Results
 Discussion
 Materials and Methods
 References
 
Cellular senescence, also known as replicative senescence, is a process during which cells lose their proliferative potential after a limited number of population doublings (PDs). Senescence is accompanied by a specific set of changes in morphology and in gene expression. For example, senescence-associated β-galactosidase (SA-β-gal) activity appears, the cell cycle is irreversibly arrested at the G1 phase and expression of cyclin-dependent kinase inhibitor (CDKI) increases (Hayflick, 1965Go; Wong and Riabowol, 1996Go). In addition, CDK4 and CDK6 can be inhibited by p16INK4{alpha} (CDKN2A), an important cell-cycle inhibitor that can induce senescence (Collins and Sedivy, 2003Go). The p16INK4{alpha} protein is thought to be an important biomarker of aging in vivo (Krishnamurthy et al., 2004Go) because its accumulation can trigger the onset of cellular senescence (Duan et al., 2001Go).

Caloric restriction (CR) increases the lifespan of many organisms. Some studies indicate that SIRT1 might mediate the effects of CR by repressing peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) activity (Picard et al., 2004Go). PPAR{gamma} is a member of the nuclear receptor superfamily of ligand-activated transcription factors. PPAR{gamma} plays an important role the induction of cellular differentiation and the inhibition of cell growth by promoting cell-cycle arrest (Chang and Szabo, 2000Go; Elstner et al., 1998Go; Kubota et al., 1998Go; Mueller et al., 1998Go; Sarraf et al., 1998Go; Tontonoz et al., 1997Go). Guan et al. reported that PPAR{gamma} activation induces the expression of p16INK4{alpha} and is accompanied by G1 arrest (Guan et al., 1999Go). Gizard et al. also demonstrated that PPAR{alpha}, another member of the receptor superfamily to which PPAR{gamma} belongs, can bind to the p16 promoter and increase p16INK4{alpha} expression (Gizard et al., 2005Go). These data suggest that PPAR{gamma} can accelerate cellular senescence by regulating p16INK4{alpha} expression. However, a role for PPAR{gamma} in senescence and the mechanisms by which PPAR{gamma} regulates p16INK4{alpha} remain poorly understood. In the present study, we demonstrate a role for PPAR{gamma} in cellular senescence. Moreover, we identify the molecular mechanisms by which PPAR{gamma} regulates the expression of p16. Our results indicate that PPAR{gamma} dephosphorylation might play an important role in cellular senescence.


   Results
Top
 Summary
 Introduction
 Results
Discussion
 Materials and Methods
 References
 
Sustained PPAR{gamma} overexpression induced premature senescence and irreversible cell-growth arrest
The senescent state of normal human diploid fibroblast cells is characterized by enlarged, flattened cells with prominent lipofuscin granules and irreversible growth arrest (Hayflick and Moorhead, 1961Go). To determine the effects of PPAR{gamma} expression on cellular senescence, young 2BS (PD25) and WI-38 (PD20) cells were transfected with the expression plasmids pcDNA3.1 and pcDNA-PPAR{gamma} (termed vector and PPAR{gamma}, respectively). After sustained selection with G418, the transformants were obtained. Western blot results (Fig. 1A) confirmed that PPAR{gamma} overexpression significantly increased the expression of the wild-type PPAR{gamma} in 2BS and WI-38 cells. The transformants were analyzed for the relative senescence markers. Untransfected young, middle-aged and senescent 2BS cells were also analyzed for comparison (see supplementary material Fig. S1). To further analyze the effect of PPAR{gamma} activation on senescence, the influence of the selective PPAR{gamma} agonists troglitazone (20 µM) and pioglitazone (10 µM) (see supplementary material Fig. S1), which have reduced activity towards PPAR{alpha} or PPARβ (Willson et al., 1996Go), on senescence markers was also tested.


Figure 1
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  		Fig. 1. Ligand-activated PPAR{gamma} 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{gamma} (PPAR{gamma}) 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{gamma} overexpression in PPAR{gamma}-transfected cells compared with vector-transfected cells. Western blotting was performed using specific antibodies against PPAR{gamma} as indicated. The β-actin lane serves as a loading control. (B) Vector-transfected and PPAR{gamma}-transfected cells were stained for SA-β-gal activity (blue), a classical marker of senescence. (C) Growth curves of vector-transfected and PPAR{gamma}-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{gamma}-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.). *P<=0.05 vs vehicle-treated vector cells; #P<=0.001 vs vehicle-treated vector cells.

 

PPAR{gamma} activation causes a senescence-like cell morphology and increased SA-β-gal activity
The specific senescence-associated marker pH 6.0 optimum β-galactosidase (SA-β-gal) was assayed by X-gal staining. Virtually all treated PPAR{gamma} cells (PD42) were strongly stained blue, with gross enlargement and flattened morphology resembling senescent cells (see supplementary material Fig. S1A). However, no significant morphological changes were observed in untreated vectors (PD42), which retained a refractive cytoplasm with long thin projections, and only a few dispersed cells were SA-β-gal-stained (Fig. 1B). For untreated PPAR{gamma} and treated vector cells (all at PD42), the positive ratio was comparable with that of middle-aged cells (PD42) (see Fig. 1B and supplementary material Fig. S1A). To determine whether the effect of PPAR{gamma} is a general accompaniment to senescence, we extended our study to investigate other normal human diploid fibroblast WI-38 cells. We obtained similar results (Fig. 1B).

The activation of PPAR{gamma} leads to growth inhibition
To observe the impact of PPAR{gamma} on cell proliferation, the growth curves for vector and PPAR{gamma} cells (all at PD42) were compared. The curve of treated PPAR{gamma} cells approached that of senescent cells (PD62), showing near complete inhibition of growth (see Fig. 1C and supplementary material Fig. S1B). Untreated PPAR{gamma} and treated vector cells had almost the same growth rate or growth potential as middle-aged cells (PD42); by contrast, untreated vector cells had stronger growth potential than untreated PPAR{gamma} and treated vector cells (see Fig. 1C and supplementary material Fig. S1B).

PPAR{gamma} activation accelerated G1 cell-cycle arrest
To clarify the mechanisms underlying growth-rate inhibition, the cell-cycle profile was analyzed by flow cytometry. Each experiment was performed at least three times and representative data are shown in Fig. 1D. Flow-cytometry assay revealed that PPAR{gamma} overexpression or agonist treatment increased the proportion of 2BS cells in the G0-G1 phases (P<0.05 vs vehicle-treated vector cells), and the greatest increase was observed when cells were both transfected with PPAR{gamma} and treated with agonist (P<0.001 vs vehicle-treated vector cells) (see Fig. 1D and supplementary material Fig. S1C). Thus, PPAR{gamma} activation might influence cell proliferation by influencing cell-cycle progress.

PPAR{gamma} activation results in a reduction of the 2BS replicative lifespan
The replicative senescence of normal human diploid fibroblasts is directly correlated to the number of PDs rather than to the growth and metabolic time (Dai and Enders, 2000Go; Hayflick, 1965Go). After completing a finite number of divisions, cells enter permanent growth arrest. To determine the effects of PPAR{gamma} overexpression on the lifespan of 2BS cells, the number of PDs for PPAR{gamma}-transfected and vector-transfected cells from the same batch of young cells was counted. The results revealed that PPAR{gamma} cells treated with troglitazone ceased cell division approximately 12 PDs earlier than did vector cells treated with troglitazone. Notably, treated PPAR{gamma} cells ceased dividing 17 PDs earlier than did untreated vector cells and 19 PDs earlier than did normal cells. Untreated PPAR{gamma} and treated vector cells had a slightly shorter lifespan than did untreated vector cells (Table 1).


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  		Table 1. Cumulative population doublings of cells transfected with vector, PPAR{gamma} or control

 

Silencing PPAR{gamma} delays a senescence-like state and induces lifespan extension
To determine the effects of PPAR{gamma} silencing on cellular senescence, young 2BS and WI-38 cells were transfected with the expression plasmids pSilencer 2.1-U6 neo and pSilencer-PPAR{gamma} (termed RNAi vector and siPPAR{gamma}, respectively). After sustained drug selection, G418-resistant cell clones were obtained. Using western blotting, we detected the expression of PPAR{gamma} in vector-transfected and siPPAR{gamma}-transfected cells. We found that, in siPPAR{gamma}-transfected cells, PPAR{gamma} levels decreased significantly relative to RNAi-vector-transfected cells (Fig. 2A). Transfected cells were then analyzed for relative senescence markers.


Figure 2
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  		Fig. 2. The silencing of PPAR{gamma} 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{gamma} (siPPAR{gamma}) 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{gamma} silencing in siPPAR{gamma}-transfected cells compared with RNAi-vector-transfected cells. Western blotting was performed using specific antibodies against PPAR{gamma} as indicated. The β-actin lane serves as a loading control. (B) RNAi-vector-transfected and siPPAR{gamma}-transfected cells were stained for SA-β-gal activity (blue), a classical marker of senescence. (C) Growth curves of RNAi-vector-transfected and siPPAR{gamma}-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{gamma}-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.). *P<=0.05 vs vehicle-treated RNAi-vector cells.

 
PPAR{gamma} silencing inhibits SA-β-gal activity
No SA-β-gal activity was observed in treated and untreated siPPAR{gamma} cells (all at PD51), whereas treated RNAi-vector cells (PD51) were strongly stained blue. Only sporadic SA-β-gal-positive cells were observed in untreated RNAi vectors (PD51) (Fig. 2B). We obtained similar results with WI-38 cells (Fig. 2B).

siPPAR{gamma} promotes cell growth
The impact of PPAR{gamma} gene-specific silencing on cell growth was evaluated. The curve of treated and untreated siPPAR{gamma} cells (all at PD51) advanced quickly, indicative of a strong proliferation potential. Control-treated and untreated RNAi-vector cells (all at PD51) grew slower than did the siPPAR{gamma} cells. Moreover, untreated RNAi-vector cells grew slightly faster than did the treated RNAi-vector cells (Fig. 2C).

siPPAR{gamma} postponed G1 cell-cycle arrest
Treated and untreated siPPAR{gamma} cells (all at PD51) exhibited significantly postponed irreversible growth arrest, whereas treated and untreated RNAi-vector cells (all at PD51) exhibited an increased proportion of 2BS cells in the G0-G1 phases. Furthermore, treated RNAi-vector cells had a slightly higher percentage of cells in the G0-G1 phases than did the corresponding untreated RNAi-vector cells (Fig. 2D).

siPPAR{gamma} results in a finite extension of 2BS replicative lifespan
To determine the effects of PPAR{gamma} gene-specific silencing on the lifespan of 2BS cells, the number of PDs for siPPAR{gamma}-transfected and RNAi-vector-transfected cells from the same batch of young cells was counted. The lifespan of treated siPPAR{gamma} cells was about 17 PDs longer than treated RNAi-vector cells and about 13 PDs longer than untreated RNAi-vector cells. Treated RNAi-vector cells had a slightly shorter lifespan than did the untreated RNAi-vector cells. No significant lifespan extension was observed in untreated siPPAR{gamma} cells or treated siPPAR{gamma} cells (Table 2). Taken together, these findings indicated that PPAR{gamma} activation could weaken the replicative capacity, reduce the replicative lifespan, and ultimately promote the onset of senescence of 2BS and WI-38 cells, whereas RNAi-mediated silencing of PPAR{gamma} gene exerted the opposite effects.


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  		Table 2. Cumulative population doublings of cells transfected with RNAi vector or siPPAR{gamma}

 

PPAR{gamma}-mediated regulation of p16INK4{alpha} expression
Previous work reported that PPAR{gamma} activation induces the expression of p16INK4{alpha} (Guan et al., 1999Go) and that accumulation of p16INK4{alpha} triggers the onset of cellular senescence (Duan et al., 2001Go). Therefore, we hypothesized that p16 is a PPAR{gamma} target gene. First, we determined whether PPAR{gamma} overexpression could upregulate p16 protein expression. Western blot analysis revealed that PPAR{gamma} overexpression or troglitazone treatment enhanced p16 protein levels in 2BS and WI-38 cells, and the greatest increase was observed when cells were both overexpressed with PPAR{gamma} and treated with troglitazone (Fig. 3A). To further verify this finding, we silenced the PPAR{gamma} gene and examined the p16INK4{alpha} protein levels in 2BS and WI-38 cells. Western blot assays revealed markedly reduced p16INK4{alpha} expression in the siPPAR{gamma}-transfected cells compared with RNAi-vector-transfected cells. Furthermore, PPAR{gamma}-agonist treatment did not increase p16INK4{alpha} levels (Fig. 3B). To determine the specificity and efficiency of siPPAR{gamma}, we detected the expression of related proteins in RNAi-vector-transfected, negative-transfected [the expression plasmids pSilencer-negative control small interfering RNA (siRNA) with sequences that have no homology to any known mammalian gene, termed as negative] and siPPAR{gamma}-transfected cells by western blotting. Our findings show that, in siPPAR{gamma}-transfected cells, the PPAR{gamma} level reduced markedly compared with RNAi-vector- and negative-transfected cells. However, the levels of PPAR{alpha} and PPARβ in siPPAR{gamma}-transfected cells were similar to RNAi-vector- and negative-transfected cells (see supplementary material Fig. S2A). These data identified the specificity and efficiency of siPPAR{gamma}. Western blot assays revealed a decreased p16INK4{alpha} expression in siPPAR{gamma}-transfected cells compared with negative-transfected cells (see supplementary material Fig. S2B). The results were consistent with Fig. 3B, which confirmed further that the knockdown of PPAR{gamma} caused a reduction in p16INK4{alpha} expression. These results suggest that PPAR{gamma} activation upregulates the expression of p16INK4{alpha}.


Figure 3
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  		Fig. 3. PPAR{gamma} 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{gamma} (PPAR{gamma}), pSilencer 2.1-U6 neo (RNAi vector) or pSilencer-PPAR{gamma} (siPPAR{gamma}) and treated, as indicated, with 20 µM troglitazone or DMSO (vehicle) for 72 hours. (A) Western blot analysis of PPAR{gamma} and p16INK4{alpha} expression in vector-transfected or PPAR{gamma}-transfected cells. (B) Western blot analysis of PPAR{gamma} and p16INK4{alpha} expression in RNAi-vector-transfected or siPPAR{gamma}-transfected cells. Western blotting was performed using specific antibodies against PPAR{gamma} and p16INK4{alpha} as indicated. The β-actin lane served as a loading control.

 
PPAR{gamma} binds to the PPRE-containing region of the endogenous p16 promoter
We examined in vivo DNA-binding activity of PPAR{gamma} to the p16 promoter. A peroxisome proliferator response element (PPRE) has been identified in the p16 promoter sequence (Fig. 4A) and is located at position –1023 relative to the translation initiation site (Gizard et al., 2005Go). We found that PPAR{gamma} bound the PPRE both in young cells and in senescent cells, and both young and senescent cells had similar amplification without troglitazone stimulation (Fig. 4B). However, the binding of PPAR{gamma} was more robust in senescent cells than in young cells after stimulation with troglitazone or pioglitazone (Fig. 4B,D). Real-time PCR can increase the accuracy and precision of chromatin immunoprecipitation (ChIP) measurements, allowing for the detection of changes of less than twofold (Johnson and Bresnick, 2002Go). Quantitative real-time PCR analyses revealed that, after treatment with PPAR{gamma} agonists, there was a significant increase in the binding activity of PPAR{gamma} in both young and senescent 2BS cells, but the magnitude of change was much greater in senescent 2BS cells (Fig. 4C,E). Following the addition of GW9662, a PPAR{gamma} antagonist (Bendixen et al., 2001Go; Wright et al., 2000Go), the binding of PPAR{gamma} was weaker in senescent cells than in young cells (Fig. 4F). These results indicate that PPAR{gamma} binds to the p16 promoter in vivo and imply that the role of PPAR{gamma} in p16 transcriptional regulation is ligand dependent. As negative controls, PCR amplification using primers covering a region located immediately downstream of the –1023 site (PPRE) failed to yield a significant signal (Fig. 4G); neither the irrelevant antibody control (β-actin) nor the negative control (no antibody sample) had amplification products. Taken together, these results demonstrated the specificity of PPAR{gamma} immunoprecipitation and PCR amplification.


Figure 4
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  		Fig. 4. PPAR{gamma} binds to the PPRE-containing region in the p16 gene promoter. (A) Schematic diagram of the p16 gene promoter. `PPRE' denotes putative PPAR{gamma}-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. *P<=0.05. (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{gamma} 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.

 

Ligand-activated PPAR{gamma} induces transcription of p16
Subsequently, we analyzed the effects of PPAR{gamma} on the expression of the p16 reporter gene, in which the luciferase gene is driven by a p16 promoter. 2BS cells were transfected with various combinations of plasmids that expressed pcDNA3.1, pcDNA-PPAR{gamma}, pSilencer 2.1-U6 neo or pSilencer-PPAR{gamma} together with wild-type and mutant p16-Luc (luciferase). Cells were then treated with troglitazone (20 µM), pioglitazone (10 µM) or GW9662 (10 µM). Reporter activity was enhanced by treatment with PPAR{gamma} agonists, and this activity was higher in cell lysates that contained PPAR{gamma} from senescent cells (about 13-fold) than in those that contained PPAR{gamma} from young cells (about fourfold) (Fig. 5A). In WI-38 cells, reporter activity was also higher in cell lysates that contained PPAR{gamma} from senescent cells (about tenfold) than in those that contained PPAR{gamma} from young cells (about sixfold) (Fig. 5A). Mutation of p16-Luc or siPPAR{gamma} resulted in a significant, albeit incomplete, reduction of p16 promoter activation (Fig. 5A,B). Identical results were obtained in cells treated with GW9662 (10 µM) (Fig. 5A). Taken together, these data suggest that ligand-activated PPAR{gamma} induced transcriptional activity of the p16 promoter, and this induction was stronger in senescent cells than in young cells.


Figure 5
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  		Fig. 5. Induction of human p16 promoter activity by ligand-activated PPAR{gamma}. (A) 2BS and WI-38 cells were transfected with expression plasmids pcDNA3.1 (vector) or pcDNA-PPAR{gamma} (PPAR{gamma}) 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{gamma} (siPPAR{gamma}) 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. *P<=0.05.

 

Silencing the p16 gene by RNA interference
In order to further verify that PPAR{gamma} could promote senescence via p16INK4{alpha}, we evaluated the role of p16 in mediating the senescence effects of PPAR{gamma} activation in 2BS cells. Young 2BS cells (PD25) were transfected with the expression plasmids pSilencer 2.1-U6 neo and pSilencer-p16 (termed RNAi vector and sip16, respectively). After sustained selection with G418, the transformants (all at PD51) were obtained. Western blot results revealed that, in sip16-transfected cells, p16 levels decreased significantly relative to RNAi-vector-transfected cells (Fig. 6A). It is noteworthy that, similar to what occurred in siPPAR{gamma}-transfected cells, the SA-β-gal-positive staining ratio was drastically decreased in treated and untreated sip16-transfected cells (Fig. 6B, Fig. 2B). Interestingly, sip16-transfected cells proliferated at a much higher rate than did RNAi-vector-transfected cells. Moreover, sip16-transfected cells did not respond to troglitazone treatment (Fig. 6C). Accordingly, the ratios of cells in G0-G1 phases were reduced in sip16-transfected cells and were not affected by troglitazone treatment (Fig. 6D). Altogether, these data indicate that the silencing of p16INK4{alpha} caused resistance to PPAR{gamma}-agonist-induced senescence.


Figure 6
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  		Fig. 6. The silencing of p16 in 2BS cells prevented the appearance of senescence-associated features and the growth-inhibition effects of PPAR{gamma} 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{alpha} 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.). *P<=0.05 vs vehicle-treated RNAi-vector cells.

 

PPAR{gamma} expression in young and senescent cells
Because PPAR{gamma} DNA-binding activity and transcriptional activity increased as cells approached the end of their replicative lifespan in culture, we further evaluated the expression patterns of PPAR{gamma} during successive passages. We therefore measured endogenous mRNA and protein expression levels of PPAR{gamma} and p16INK4{alpha}. Using reverse transcription-polymerase chain reaction (RT-PCR), PPAR{gamma} mRNA, p16 mRNA and GAPDH mRNA levels were analyzed in young (PD25), middle-aged (PD42) and senescent (PD62) 2BS cells. Our findings demonstrate that the levels of PPAR{gamma} mRNA were similar in young, middle-aged and senescent cells, whereas the p16 mRNA levels were increased in senescent cells relative to other groups (Fig. 7A). In addition, western blot analysis was performed on the 2BS and WI-38 cells. Protein levels of PPAR{gamma} did not change appreciably as cells aged, whereas p16INK4{alpha} protein levels increased as cells aged in culture (Fig. 7B).


Figure 7
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  		Fig. 7. The expression level of PPAR{gamma}. (A) The relative amounts of p16, PPAR{gamma} 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{gamma} or p16 primers. GAPDH was used as an internal control for normalization purposes. (B) Western blot analysis of PPAR{gamma} and p16INK4{alpha} expression in 2BS and WI-38 cells. Total proteins were extracted, and western blotting was performed using specific antibodies against PPAR{gamma} and p16INK4{alpha} as indicated. The β-actin lane serves as a loading control.

 
Phosphorylation of PPAR{gamma} represses its transactivating function
The above findings indicate that PPAR{gamma} levels remain constant in young and senescent cells, but they do not explain why the transcriptional activity of PPAR{gamma} increased in senescent cells. Phosphorylation modifications reportedly affect PPAR{gamma} transcriptional activity (Hu et al., 1996Go; Lazennec et al., 2000Go; Han et al., 2000Go); therefore, we analyzed the phosphorylation state of PPAR{gamma} in young and senescent 2BS and WI-38 cells. We found that the phosphorylation level of PPAR{gamma} decreased as cells aged (Fig. 8A). To evaluate the effect of PPAR{gamma} phosphorylation on its transcriptional function, we created a mutant of pcDNA-PPAR{gamma}, in which the serine at position 84, the potential phosphorylation site, was changed to alanine [pcDNA-PPAR{gamma} (S84A)]. We then analyzed the effects of PPAR{gamma} phosphorylation on the expression of the p16 reporter gene in which the luciferase gene is driven by the p16 promoter. 2BS cells were transfected with various combinations of plasmids that expressed pcDNA3.1(–), pcDNA-PPAR{gamma} (WT) or mutant pcDNA-PPAR{gamma}(S84A) together with wild-type p16-Luc. Cells were then treated with troglitazone (20 µM) or pioglitazone (10 µM). The results revealed that the untreated mutant PPAR{gamma} (S84A) exhibited greater transactivation (about 4.5-fold) than untreated wild-type PPAR{gamma} (about twofold). Furthermore, the treated S84A mutant activated luciferase expression to a significantly greater extent (about 6.5-fold) than treated wild-type PPAR{gamma} (about threefold). Our finding indicated that the S84A mutant showed a high level of transactivation, comparable with that of wild-type PPAR{gamma} (Fig. 8B). Taken together, these data suggest that phosphorylation of PPAR{gamma} represses its transcriptional activity.


Figure 8
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  		Fig. 8. Phosphorylation represses the transactivating function of PPAR{gamma}. (A) Western blot analysis of phosphorylation levels of PPAR{gamma} in young and senescent 2BS and WI-38 cells. Total proteins were extracted, and western blotting was performed using specific antibodies against PPAR{gamma} and phosphorylated PPAR{gamma} (p-PPAR{gamma}) as indicated. Arrow indicates phosphorylated PPAR{gamma}, and the observed doublet is reported as two translation initiation sites. (B) 2BS cells were transfected with expression plasmids pcDNA3.1 (–), pcDNA-PPAR{gamma} (WT) or mutant pcDNA-PPAR{gamma} (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. *P<=0.05.

 


   Discussion
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 Results
 Discussion
Materials and Methods
 References
 
Senescence is the state or process of aging at the cellular level, and is thought to relate to age-related diseases and tumorigenesis (Campisi, 2001Go; Campisi, 2005Go; Weinstein and Ciszek, 2002Go). Senescence may be described as accumulated DNA damage, a limited number of cell divisions and a decreased ability to remove free radicals (Weinstein and Ciszek, 2002Go). CR has been proposed to extend lifespan and slow aging. Some studies suggest that SIRT1, the primary molecule mediating the effects of CR, functions by repressing PPAR{gamma} activity (Picard et al., 2004Go). PPAR{gamma} might play an important role in cellular senescence; however, the function of PPAR{gamma} in the progression of cellular senescence has never been described. In the present study, we demonstrated that PPAR{gamma} activation promoted cellular senescence (Figs 1, 2), an effect attributed to the induction of p16. p16INK4{alpha} is an important cell-cycle inhibitor, and its accumulation triggers the onset of cellular senescence (Duan et al., 2001Go). An upregulation of p16INK4{alpha} expression by PPAR{gamma} agonists provides a mechanism for senescence being repressed by CR and also a model (Fig. 9) that regulates cellular senescence.


Figure 9
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  		Fig. 9. Model of PPAR{gamma}-regulated cellular senescence.

 
The nuclear receptor superfamily of PPARs regulates the transcription of numerous target genes after dimerizing with the retinoid X receptor (RXR) and binding to PPRE (a specific DNA-binding site) (Mangelsdorf et al., 1995Go). PPREs usually consist of a direct repeat of the hexanucleotide AGGTCA sequence, separated by one or two nucleotides (DR1 or DR2) (Michalik et al., 2004Go). Recently, one report indicated that PPAR{alpha} can bind to PPRE in the p16 promoter and increase p16INK4{alpha} expression (Gizard et al., 2005Go). Because the nuclear receptor superfamily of PPARs can bind to the same DNA-binding site, PPRE, we hypothesize that PPAR{gamma} can also bind to PPRE in the p16 promoter and thereby regulate p16INK4{alpha} expression. Western blot analysis demonstrated that PPAR{gamma} overexpression and PPAR{gamma}-agonists treatment enhanced endogenous expression of p16INK4{alpha}, whereas RNA interference of PPAR{gamma} inhibited the expression of p16INK4{alpha} (Fig. 3). These results suggested that the activation of PPAR{gamma} induced the expression of p16INK4{alpha} in 2BS and WI-38 cells.

In the search for the molecular mechanisms involved in the regulation by PPAR{gamma} of p16, PPAR{gamma} activation was found to upregulate p16 promoter activity, thus identifying for the first time a molecular mechanism by which PPAR{gamma} directly interferes with senescence progression. We observed that both DNA-binding activity and transcriptional activity of PPAR{gamma} were increased several fold in senescent cells, and the increased activity is ligand-dependent (Figs 4, 5).

2BS cells were previously isolated from human fetal lung fibroblast tissue and have been fully characterized (Tang et al., 1994Go). The maximum population doubling of human diploid fibroblasts is limited in culture, so they are widely used as a model of cellular senescence. Previous reports have suggested that fibroblast cellular senescence occurs as a consequence of a `genetic program' (Goldstein, 1990Go). This program has been partially characterized by gene expression patterns during the progression of successive passages. Sasaki et al. reported that PPAR{gamma} mRNA levels do not significantly differ by sex or age in lung tissue (Sasaki et al., 2002Go). However, Ye et al. found that the expression of PPAR{gamma} at both the mRNA and protein level in adipose tissue of older rats was dramatically decreased and, likewise, that the expression of PPAR{gamma} mRNA in omental adipose tissue of elderly men was significantly decreased (Ye et al., 2006Go). These conflicting data regarding PPAR{gamma} expression patterns might be due to the various roles of PPAR{gamma} in different tissues during the progression of successive passages. Because PPAR{gamma} activation increases during aging, fat-deposit size reportedly declines and lipids are redistributed to muscle, bone marrow and other tissues (Moerman et al., 2004Go; Kirkland et al., 2002Go). Our results indicate that the mRNA and protein level of PPAR{gamma} in young cells is similar to that in senescent cells (Fig. 7). We found that PPAR{gamma} activity increased in senescent cells, although the expression of PPAR{gamma} did not appear to be upregulated.

Research has demonstrated that PPAR{gamma} activity is modulated by phosphorylation, which inhibits PPAR{gamma} transcriptional activity (Hu et al., 1996Go; Lazennec et al., 2000Go; Han et al., 2000Go). To determine why the transcriptional activity of PPAR{gamma} increases as cells age, we analyzed the phosphorylation state of PPAR{gamma} in both young and senescent 2BS and WI-38 cells. As expected, the levels of PPAR{gamma} phosphorylation decreased as cells aged (Fig. 8A). We also found that mutant PPAR{gamma} (mutation of serine 84 to alanine, which abolished phosphorylation of PPAR{gamma}) has higher transcriptional activity than wild-type PPAR{gamma} (Fig. 8B). These results indicate that phosphorylation of PPAR{gamma} represses the transcriptional activity of PPAR{gamma} itself. Three possible mechanisms may explain the modulation of PPAR{gamma} transactivation activity: (a) phosphorylation of PPAR{gamma} might decrease its affinity for its cognate ligand; (b) the phosphorylation of PPAR{gamma} could influence its interactions with co-repressors and/or coactivators of transcription; and (c) phosphorylation of PPAR{gamma} might promote its degradation by the ubiquitin-proteasome system in response to ligand activation. According to the first mechanism, hyperphosphorylated PPAR{gamma} should exhibit decreased ligand binding. Our results indicated that the DNA-binding activity and transcriptional activity of PPAR{gamma} increased upon agonist treatment and these changes were much greater in senescent cells than that in young cells (Figs 4, 5). This indicated that, in young cells, hyperphosphorylation of PPAR{gamma} decreases its transcriptional activity. However, hyperphosphorylation of PPAR{gamma} does not alter its DNA-binding activity, because the DNA-binding activity of PPAR{gamma} is similar in untreated young cells and untreated senescent cells (Fig. 4). The second pattern is currently under investigation in our laboratory. Finally, because PPAR{gamma} levels remained constant during successive passages (Fig. 7), we hypothesize that the third mechanism is not involved in PPAR-regulated senescence in 2BS and WI-38 cells.

p16INK4{alpha} is an important cell-cycle inhibitor that can induce senescence and repress tumor cell growth (Collins and Sedivy, 2003Go). Its loss or inactivation is correlated with cell immortality (Ruas and Peters, 1998Go). Although p16INK4{alpha} plays an important role in cellular senescence and tumorigenesis, its transcriptional control is poorly understood. Here, we report, for the first time to our knowledge, the molecular mechanism by which PPAR{gamma} upregulates the expression of p16INK4{alpha} in 2BS and WI-38 cells. Our results, which show that p16INK4{alpha} is required for PPAR{gamma}-agonist-induced senescence (Fig. 6), verify this assertion. However, recent studies have proposed p16 regulatory pathways that are distinct from those described here. Ohtani et al. proposed a model in which the upregulation of p16INK4{alpha} depended on the accumulation of Ets1 and the absence of interference by Id1 during senescence (Ohtani et al., 2001Go). Zheng et al. found that Id1 regulated p16INK4{alpha} levels through interactions with E47 (Zheng et al., 2004Go). Wang et al. reported that a 24-kDa protein might inhibit the expression of p16INK4{alpha} by interacting with the INK4{alpha} transcription silence element (ITSE) (Wang et al., 2001Go). These findings do not contradict the present conclusions. It is clear that the p16 promoter is subject to multiple levels of control (Hara et al., 1996Go; Jacobs et al., 1999Go); therefore, p16 regulation cannot be explained by a single isolated pathway.

In summary, in senescent cells, dephosphorylation of PPAR{gamma} increases its transcriptional activity. The increased PPAR{gamma} activity might be one reason for the elevated p16INK4{alpha} expression in senescent 2BS and WI-38 cells, which in turn contributes to the onset of cellular senescence (Fig. 9). Various polyunsaturated fatty acids, the major dietary constituents, are specific ligands for PPAR{gamma} (Forman et al., 1995Go; Kahn et al., 2000Go). Much research shows that fatty acids can influence the transcriptional activity of PPAR{gamma}. In our study, PPAR{gamma} accelerated senescence by inducing p16INK4{alpha} expression in a ligand-dependent manner; therefore, PPAR{gamma} might underlie a key switch between exterior factors (such as diet) and interior factors (such as the p16 gene). Finally, we demonstrated a gene-environment effect induced by PPAR{gamma}-agonist administration, which results in senescence-like growth arrest with p16INK4{alpha} expression. The effect was more marked in senescent cells than in young cells. Our study might offer an opportunity to investigate gene-environment interactions associated with cellular senescence in health and disease.


   Materials and Methods
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 Summary
 Introduction
 Results
 Discussion
 Materials and Methods
References
 
Antibodies, reagents and plasmids
Antibodies against PPAR{gamma} (SC-7273), PPAR{alpha} (SC-9000), PPARβ (SC-1983) and β-actin (SC-1616) were purchased from Santa Cruz Biotechnology. Anti-phospho-PPAR{gamma} (05-816) and anti-p16 (MS-887-P1) antibodies were purchased from Upstate. Pioglitazone and GW9662 were purchased from Cayman. Troglitazone was purchased from Calbiochem or Cayman. The PPAR{gamma} expression plasmid cloned in pcDNA3.1 was constructed as described previously (Fu et al., 2001Go). p16 cDNA in pBluescript was a kind gift from David Beach (Howard Hughes Medical Institute, Cold Spring Harbor, NY). The full-length p16 cDNA (800 bp) was placed into the expression vector pcDNA3.1 in both orientations. A p16-promoter fragment that contained 1040 bp was obtained via PCR from the 3070-bp pGL2-Basic vector, which was generously provided by Gorden Peters (Imperial Cancer Research Fund Laboratories, London, UK).

siRNA preparation
The siRNA was designed as reported previously. The sequence of the sense strand of PPAR{gamma} siRNA was 5'-GCCCTTCACTACTGTTGAC-3' (Kelly et al., 2004Go); p16 siRNA was 5'-AGAACCAGAGAGGCTCTGA-3' (Zhou et al., 2004Go); and negative control siRNA was 5'-TTCTCCGAACGTGTCACGT-3' (Genechem). The hairpin-siRNA template oligonucleotides were chemically synthesized with 5'-phosphate, 3'-hydroxyl, and two base overhangs on each strand. Then the template oligonucleotides were inserted into the BamHI and HindIII sites of the pSilencer 2.1-U6 neo vector.

Cell culture and transfection
Human embryonic lung diploid fibroblast 2BS cells (obtained from the National Institute of Biological Products, Beijing, China) were previously isolated from female fetal lung fibroblast tissue and have been fully characterized (Tang et al., 1994Go). The current expected lifespan is approximately PD70. 2BS cells are considered to be young at PD30 or below and to be fully senescent at PD55 or above. Human embryonic lung diploid fibroblast WI-38 cells (ATCC number: CCL75) were obtained from the Chinese Academy of Sciences (Shanghai, China). The current expected lifespan is approximately PD55. WI-38 cells are considered to be young at PD25 or below and to be fully senescent at PD50 or above. Cells were maintained in Dulbecco's modified Eagle's medium (Gibco) supplemented with 10% fetal bovine serum (FBS, Hyclone), 100 units/ml penicillin, and 100 µg/ml streptomycin at 37°C in 5% CO2.

Young cells were grown to 80-90% confluence. Expression plasmids were transfected with Lipofectin reagent (Life Technologies) according to the manufacturer's instructions. Pools of stable transformants were obtained by sustained selection of 300 µg/ml G418 (Life Technologies). PDs were calculated with the formula PD=log(n2/n1)/log2, where n1 is the number of cells seeded and n2 is the number of cells recovered (Shay and Wright, 1989Go).

Growth curves
Cells were detached and seeded into 96-well plates, with 2000 cells per well. After overnight incubation, cells were treated with 20 µM troglitazone or 10 µM pioglitazone diluted in DMSO (Sigma). All cells received identical volumes of DMSO and were exposed to each drug for 6 days; medium and drug were changed every 48 hours. At the indicated times, cells were stained with 20 µl 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT, 10 mg/ml in PBS; Sigma) for 3 hours and then dissolved with DMSO. The optical density at 570 nm was determined.

Cell-cycle analysis and synchronization
When cells reached 70-80% confluence, they were washed with PBS, detached with 0.25% trypsin and fixed with 75% ethanol overnight. After treatment with 1 mg/ml RNase A (Sigma) at 37°C for 30 minutes, cells were resuspended in 0.5 ml of PBS and stained with propidium iodide in the dark for 30 minutes; DNA contents were measured by fluorescence-activated cell sorting on a FACScan flow cytometry system (BD Biosciences). The data were analyzed using CellFiT software.

For synchronization, 2BS cells were rendered quiescent by serum deprivation for 48 hours and then stimulated to re-enter the cell cycle by the addition of serum to a final concentration of 10%. G1-phase cells were harvested at 8 hours after serum stimulation.

SA-β-gal staining
Cells were washed twice in PBS, fixed for 3-5 minutes at room temperature in 3% formaldehyde and washed with PBS again. Then cells were incubated overnight at 37°C without CO2 in a freshly prepared staining solution [1 mg/ml 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal), 40 mM citric acid/sodium phosphate, pH 6.0, 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 150 mM NaCl, 2 mM MgCl2] (Dimri et al., 1995Go). At least 200 cells were counted in randomly chosen fields from each culture well.

Western blotting
Cells were lysed in modified radioimmune precipitation assay (RIPA) buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.25% Na-deoxycholate, 1% NP-40, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, 0.2 mM sodium orthovanadate, 1 mM NaF). Protein concentration of each sample was determined by BCA protein assay reagent (Pierce); 100-150 µg of protein was electrophoresed on 15% SDS-polyacrylamide gel and transferred to nitrocellulose membrane (Millipore). The membrane was blocked and then incubated with the primary antibody in 5% non-fat dry milk in TBST (10 mM Tris-Cl, pH 7.5, 150 mM NaCl, 0.05% Tween 20) overnight at 4°C. After washing, the blots were incubated with secondary antibody conjugated to horseradish peroxidase (Amersham Biosciences) at 1:40,000 in TBST for 1 hour at room temperature. Proteins were visualized with Chemiluminescent Substrate (Pierce) according to manufacturer's instructions.

Site-directed mutagenesis
Substitution mutations were generated using the QuikChange mutagenesis kit (Stratagene) according to the manufacturer's instructions. Synthetic oligonucleotides that contained the desired bases were used in the mutagenesis. The sequence of mutations for nucleotides at positions –1023 and –1022 (GG-CC) of the 5'-flanking region of the p16 promoter was 5'-GTGTGAACCAGACAGGACAGTATTT-3' (GG-CC mutation underlined) (Gizard et al., 2005Go); mutant PPAR{gamma} with the substitution of serine-84 to alanine was 5'-GTGGAGCCTGCAGCTCCACCTTATTATTC-3' (TCT-GCT mutation) (Adams et al., 1997Go). DNA that incorporated the desired mutations was transformed into XL1-Blue supercompetent cells. Plasmid DNA was prepared and the presence of the mutations was confirmed by sequencing.

Chromatin immunoprecipitation
ChIPs were performed using the Chromatin Immunoprecipitation Assay kit (Upstate) according to manufacturer's instructions. For each experimental condition, 1x106 cells were used. At 48 hours before harvesting, cells were pre-treated with 20 µM troglitazone, 10 µM pioglitazone, 10 µM GW9662, or 1% DMSO (vehicle) as control. Cells were sonicated and lysates immunoprecipitated using the indicated antibodies. To amplify PPRE regions of the p16 promoter, the following sequences of the primers were used: p16 PPREs, 5'-GCACTCATATTCCCTTCCCCCCT-3'; p16 PPREa, 5'-GGAAGGACGGACTCCATTCTCAAAG-3'. Control p16 element was located immediately downstream of PPRE. The sequences of the primers used were as follows: p16 controls, 5'-GAAGCTGGTCTTTGGATCACTGTGC-3'; p16 controla, 5'-GACGGGGGAGAATTCTGCCTGT-3' (Gizard et al., 2005Go). All primers were synthesized at Sunbio Biotechnology (Beijing, China).

Real-time PCR and data analysis
Two-step real-time PCR was performed with 5 µl of DNA and 400 nM primers diluted to a final volume of 50 µl in SYBR Green Master Mix (Applied Biosystems). Accumulation of fluorescent products was monitored by real-time PCR using an Applied Biosystem 7300 real-time PCR system (Applied Biosystems). A melting curve was generated to ensure that a single peak of the predicted Tm was produced and no primer-dimer complexes were present. Single amplicon generation was verified by agarose gel electrophoresis. No PCR products were observed in the absence of template. Sequence Detector software (version 1.3.1) was used for data analysis and relative fold induction was determined by the comparative threshold cycle (CT). Fold-enrichments were determined by the method described in the Applied Biosystems User Bulletin and data analysis followed the methodology described in a recent report (Frank et al., 2001Go). Fold differences were calculated by correcting for each signal concentration with the concentration of input signal for each sample [(signal concentration)/(input concentration)]. Real-time RT-PCR data figures were generated using Microsoft Excel (Microsoft Corporation, USA).

Luciferase assay
2BS cells were plated in six-well culture plates in triplicate for each condition at an initial concentration of 2x105 cells/well. pGL2 luciferase reporter constructs driven by the indicated 1 µg p16 wild-type (p16 WT) or mutant (p16 mutant) promoter fragments were co-transfected with 3 µg pcDNA3.1 (vector), wild-type pcDNA-PPAR{gamma} (PPAR{gamma} or WT), mutant pcDNA-PPAR{gamma} (S84A), pSilencer 2.1-U6 neo (RNAi vector) or pSilencer-PPAR{gamma} (siPPAR{gamma}) expression plasmid. The amount of plasmid in the transfection mixture was equalized to 4 µg by adding pcDNA3.1 vector. Renilla luciferase reporter plasmid pRL-CMV (10 ng) was also co-transfected into each well as an internal control. After 24 hours, cells were treated with 20 µM troglitazone, 10 µM pioglitazone or 10 µM GW9662. Luciferase activity was assessed with a dual-luciferase reporter assay system (Promega) according to manufacturer's instructions after 48 hours of drug treatment. The enzyme activity was normalized for efficiency of transfection on the basis of Renilla luciferase activity levels and reported as relative light units (RLU). All reporter assays were performed in triplicate on at least two individual experiments and standard errors are denoted by bars in the figures.

Reverse-transcription PCR
Total RNA was isolated from 2BS cells by RNeasy kit (QIAGEN). After denaturing the total RNA at 70°C for 10 minutes, cDNA was synthesized with oligo-dT primer and reverse transcriptase (Invitrogen). PCR amplification was performed using specific primers for PPAR{gamma} as follows: PPAR{gamma}s, 5'-GAGCCCAAGTTTGAGTTTGC-3'; PPAR{gamma}a, 5'-TGGAAGAAGGGAAATGTTGG-3'. The sequences of the primers used for p16 were: p16s, 5'-CCCAACGCACCGAATAGT-3'; p16a, 5'-ATCTAAGTTTCCCGAGGTT-3'. The sequences of the primers used for GAPDH were: GAPDHs, 5'-CGAGTCAACGGATTTGGTGGTAT-3'; GAPDHa, 5'-AGCCTTCTCCATGGTGAAGAC-3'. PCR products were loaded onto an agarose gel and stained with ethidium bromide.

Statistical analysis
The data are reported as mean ± s.d. of the indicated number of experiments. Values were assessed by pairwise (one-way analysis of variance, ANOVA). In all cases, P<=0.05 and P<=0.01 was considered significant.


   Acknowledgments
 
We thank Wengong Wang for helpful discussions. This work was supported by grants from the National Basic Research Programs of China (No. 2007CB507400) and the National Natural Science Foundation of China (No. 30671064).


   Footnotes
 
Supplementary material available online at http://jcs.biologists.org/cgi/content/full/121/13/2235/DC1


   References
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 Summary
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

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R. Zhou, L. Han, G. Li, and T. Tong
Senescence delay and repression of p16INK4a by Lsh via recruitment of histone deacetylases in human diploid fibroblasts
Nucleic Acids Res., August 1, 2009; 37(15): 5183 - 5196.
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