Elimination of p19ARF-expressing cells enhances pulmonary function in mice
Michihiro Hashimoto, … , Mitsuo Maruyama, Masataka Sugimoto
Michihiro Hashimoto, … , Mitsuo Maruyama, Masataka Sugimoto
Published August 4, 2016
Citation Information: JCI Insight. 2016;1(12):e87732. https://doi.org/10.1172/jci.insight.87732.
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Research Article Aging Cell biology

Elimination of p19ARF-expressing cells enhances pulmonary function in mice

Abstract

Senescent cells accumulate in many tissues as animals age and are considered to underlie several aging-associated pathologies. The tumor suppressors p19ARF and p16INK4a, both of which are encoded in the CDKN2A locus, play critical roles in inducing and maintaining permanent cell cycle arrest during cellular senescence. Although the elimination of p16INK4a-expressing cells extends the life span of the mouse, it is unclear whether tissue function is restored by the elimination of senescent cells in aged animals and whether and how p19ARF contributes to tissue aging. The aging-associated decline in lung function is characterized by an increase in compliance as well as pathogenic susceptibility to pulmonary diseases. We herein demonstrated that pulmonary function in 12-month-old mice was reversibly restored by the elimination of p19ARF-expressing cells. The ablation of p19ARF-expressing cells using a toxin receptor-mediated cell knockout system ameliorated aging-associated lung hypofunction. Furthermore, the aging-associated gene expression profile was reversed after the elimination of p19ARF. Our results indicate that the aging-associated decline in lung function was, at least partly, attributed to p19ARF and was recovered by eliminating p19ARF-expressing cells.

Authors

Michihiro Hashimoto, Azusa Asai, Hiroyuki Kawagishi, Ryuta Mikawa, Yuji Iwashita, Kazuki Kanayama, Kazushi Sugimoto, Tadashi Sato, Mitsuo Maruyama, Masataka Sugimoto

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Figure 1

Establishment of ARF-DTR transgenic mice.

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Establishment of ARF-DTR transgenic mice. (A) A transgenic vector was co...
(A) A transgenic vector was constructed using a phage artificial chromosome, including a mouse INK4a/ARF locus. ARF exon 1β was replaced with genes encoding the diphtheria toxin receptor (DTR) (human HB-EGF I117V/L148V) fused to the self-cleaving picornavirus-derived 2A peptide sequence and firefly luciferase (DTR-Luc). (B and C) MEFs prepared from wild-type or ARF-DTR mice were cultured on a 3T3 protocol (B) or infected with control (puro) or oncogenic RasVal12-encoding retroviruses (C) in order to induce senescence. Infected cells were selected with puromycin for 3 days. Cell lysates were prepared and luciferase activity was measured. Luciferase activity was normalized to cell numbers in each sample. Values represent the mean ± SD of triplicate samples. Senescence was confirmed by the expression of p19ARF, p16INK4a, and p21 in passage 5 (P5) and Ras-expressing (Val12-expressing) MEFs. Lamin A/C was used as a loading control. (D and E) Senescence was induced in ARF-DTR MEFs by serial passages (D) or oncogenic Ras (E). MEFs were then treated with the indicated concentrations of diphtheria toxin (DT) for 24 hours, and the luciferase assay was performed. Data are representative of 2 independent experiments. Values are shown as the mean ± SD of triplicate samples. (F and G) Senescence was induced in ARF-DTR MEFs by serial passages (F) or oncogenic Ras (G). Senescent MEFs (P5 or Ras) were cultured in the absence or presence of DT (1 μg/ml) for 24 hours, and the expression of p19ARF and p16INK4a was analyzed by immunoblotting. Lamin A/C was used as a loading control.