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. 2015 Apr 29;6:7035.
doi: 10.1038/ncomms8035.

Ablation of the p16(INK4a) Tumour Suppressor Reverses Ageing Phenotypes of Klotho Mice

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Free PMC article

Ablation of the p16(INK4a) Tumour Suppressor Reverses Ageing Phenotypes of Klotho Mice

Seidai Sato et al. Nat Commun. .
Free PMC article

Abstract

The p16(INK4a) tumour suppressor has an established role in the implementation of cellular senescence in stem/progenitor cells, which is thought to contribute to organismal ageing. However, since p16(INK4a) knockout mice die prematurely from cancer, whether p16(INK4a) reduces longevity remains unclear. Here we show that, in mutant mice homozygous for a hypomorphic allele of the α-klotho ageing-suppressor gene (kl(kl/kl)), accelerated ageing phenotypes are rescued by p16(INK4a) ablation. Surprisingly, this is due to the restoration of α-klotho expression in kl(kl/kl) mice and does not occur when p16(INK4a) is ablated in α-klotho knockout mice (kl(-/-)), suggesting that p16(INK4a) is an upstream regulator of α-klotho expression. Indeed, p16(INK4a) represses α-klotho promoter activity by blocking the functions of E2Fs. These results, together with the observation that the expression levels of p16(INK4a) are inversely correlated with those of α-klotho throughout ageing, indicate that p16(INK4a) plays a previously unrecognized role in downregulating α-klotho expression during ageing.

Figures

Figure 1
Figure 1. Extension of maximum lifespan of klkl/kl mice by p16INK4a ablation.
(a) Representative photographs of 11-week-old mice of each genotype (n=3). (b) Kaplan–Meier plot showing survival of WT (male, n=20; female, n=20), p16−/− (male, n=21; female, n=22), klkl/kl (male, n=25; female, n=24) and p16−/− klkl/kl (male, n=25; female, n=28).
Figure 2
Figure 2. Reversing the ageing phenotypes of klkl/kl mice by p16INK4a ablation.
(a) Histological analysis of 11-week-old female WT, p16−/−, klkl/kl and p16−/− klkl/kl mice. Representative images of bone radiographs of femurs (X-P), HE of tissues indicated top and von Kossa staining (von Kossa) of the kidney for detecting ectopic calcification were shown. (b) The histograms indicate the quantitative analysis of X-ray transparency of femur (WT (n=3), p16−/− (n=3), klkl/kl (n=6) and p16−/− klkl/kl (n=3)), the mean linear intercept (Lm) in lung tissue (WT (n=9), p16−/− (n=9), klkl/kl (n=7) and p16−/− klkl/kl (n=9)), intestinal villi length (WT (n=14), p16−/− (n=7), klkl/kl (n=11) and p16−/− klkl/kl (n=3)), epidermal and subcutaneous fat layer thickness (WT (n=6), p16−/− (n=6), klkl/kl (n=8) and p16−/− klkl/kl (n=3)) and the percentages of calcified areas in kidneys (WT (n=3), p16−/− (n=3), klkl/kl (n=3) and p16−/− klkl/kl (n=3)). For graphs of X-ray transparency of femur and Lm in lung tissues, data were analysed by Mann–Whitney U-test and are displayed as mean±s.e.m. For graphs of intestinal villi length, epidermal and subcutaneous fat layer thickness and the percentages of calcified areas in kidneys, data were analysed by Student's t-test and are displayed as mean±s.e.m. For all graphs: *P<0.05, **P<0.01.
Figure 3
Figure 3. Bioluminescence in vivo imaging of p16INK4a expression in klkl/kl mice.
(a) Littermates of 8-week-old male p16-luc mice (WT p16-luc) and p16-luc mice homozygous for a severely downregulated hypomorphic allele of the α-klotho (klkl/kl p16-luc) were subjected to in vivo bioluminescence imaging after being incised through the throat and the anus under anaesthesia. Representative images of three independent experiments are shown (n=3 experiments). The colour bar indicates photons with minimum and maximum threshold values. (b) Bioluminescence intensity emitted from the organs was graphed (log 10 scale).
Figure 4
Figure 4. Recovery of α-klotho expression in the kidney by p16INK4a ablation.
(a) The relative levels of indicated mRNA in kidneys of 2- to 6-month-old male mice of each genotype were examined using RT–qPCR. Representative results of three individual male mice were shown. N.D. represents ‘not detected'. (b) Serum phosphate and serum calcium levels of 2- to 6-month-old male mice of each genotype were shown. Representative results of three individual mice were shown. (c) Kidneys of each genotype were subjected to western blot analysis using antibodies shown left. Calpain-1 represents the levels of activated form of Calpain-1. Vinculin was used as a loading control. Representative results of three individual male mice were shown. For all graphs, the experiments were performed in triplicate, and representative results from three independent experiments are shown. Data were analysed by Welch's t-test and are displayed as mean±s.d. *P<0.05.
Figure 5
Figure 5. Inverse correlation between α-klotho and p16INK4a expression.
(a) Immunohistochemical analysis of α-klotho (red), E-cadherin (green) and 4,6-diamidino-2-phenylindole (blue) in 11-week-old male mouse kidney sections of each genotype. Representative results of three individual male mice were shown. The histograms indicate the percentages of E-cadherin-expressing cells that were positive for α-klotho expression. At least 100 cells were scored per group. (b) The relative levels of indicated mRNA in kidneys of young (10- to 20-week-old) or old (120- to 140-week-old) Wt male mice were examined using RT–qPCR. Representative results of three individual male mice were shown. For all graphs, the experiments were performed in triplicate, and representative results from three independent experiments are shown. Data were analysed by Welch's t-test and are displayed as mean±s.d. *P<0.05, **P<0.01.
Figure 6
Figure 6. p16INK4a downregulates α-klotho gene promoter activity via E2F.
(a,b) Schematic representation of the reporter construct of mouse α-klotho gene promoter used in the analysis (left panel). The E2F-binding element is shown as a black rectangle with the sequence and firefly luciferase is shown as Luc. The reporter construct was co-transfected into mRTECs along with LacZ or Renilla plasmid. Where indicated, cells were also co-transfected with an increasing amount of p16INK4a expression plasmid (a) or with 2 μg of expression plasmid encoding E2F1 or E2F3 (b). (c) Early-passage mRTECs and kidney tissues were prepared from 6- to 10-week-old male mice and were subjected to ChIP analysis using antibodies shown at bottom and PCR primers shown at the top (red arrows). (d) The relative levels of α-klotho mRNA and protein expression in kidneys of each genotype were examined using RT–qPCR (upper panel) or western blot analysis (lower panel). Representative results of two individual 2.7-week-old male mice were shown. For all graphs, the experiments were performed in triplicate, and representative results from three independent experiments are shown. Data were analysed by Welch's t-test and are displayed as mean±s.d. *P<0.05, **P<0.01.
Figure 7
Figure 7. p16INK4a downregulates α-klotho expression in humans.
(a) The relative levels of indicated mRNA in hRTECs were examined using RT–qPCR. RNAs were prepared from early-passaged or late-passaged hRTECs (left panels), early-passaged hRTECs transfected with siRNA against p16INK4a or control (middle panels), or early-passaged hRTECs transfected with or without p16INK4a expression vector (right panels). (b) Schematic representation of the reporter construct of the human α-klotho gene promoter used in the analysis (left panel). The E2F-binding element is shown as a black rectangle with the sequence and firefly luciferase is shown as Luc. The reporter construct was co-transfected into hRTECs along with Renilla plasmid. Where indicated, cells were also co-transfected with 1.5 μg of expression plasmid encoding E2F1 or E2F3. (c) Early-passage cultured hRTECs were subjected to ChIP analysis using antibodies shown at bottom and PCR primers shown at the top (red arrows). For all graphs, the experiments were performed in triplicate, and representative results from three independent experiments are shown. Data were analysed by Welch's t-test and are displayed as mean±s.d. *P<0.05, **P<0.01.
Figure 8
Figure 8. Dual roles for the p16INK4a RB pathway in organismal ageing.
The p16INK4a has an established role in provoking cellular senescence, which is likely to cause stem cell ageing and thereby contributing to organismal ageing. Here we show that, in addition, p16INK4a also contributes to organismal ageing through blocking the expression of ageing suppressor, α-klotho.

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