Elimination of p19ARF-expressing cells enhances pulmonary function in mice
Michihiro Hashimoto, Azusa Asai, Hiroyuki Kawagishi, Ryuta Mikawa, Yuji Iwashita, Kazuki Kanayama, Kazushi Sugimoto, Tadashi Sato, Mitsuo Maruyama, Masataka Sugimoto
Michihiro Hashimoto, Azusa Asai, Hiroyuki Kawagishi, Ryuta Mikawa, Yuji Iwashita, Kazuki Kanayama, Kazushi Sugimoto, Tadashi Sato, Mitsuo Maruyama, Masataka Sugimoto
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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 3

Luciferase was expressed in alveolar fibroblasts in old ARF-DTR mice.

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Luciferase was expressed in alveolar fibroblasts in old ARF-DTR mice. (A...
(A) Lung cell fractionation. Bronchoalveolar lavage (BAL) fluid mainly containing macrophages was initially obtained from the lung. The rest of the tissue was minced, trypsinized, and seeded on a tissue culture dish. After being incubated for 1 hour, floating cells that contained epithelial cells were collected. Attached cells were trypsinized to recover alveolar fibroblasts. (B) Lysates were prepared from the collected cells, and the luciferase assay was performed. Luciferase activity was normalized to the cell number in each sample. Results of 2 independent experiments are shown. (C-E) Immunofluorescence staining of the ARF–diphtheria toxin receptor (ARF-DTR) lung. Sections were stained with antibodies against the indicated proteins. Original magnification, ×40 (C and D), ×100 (E); scale bar: 20 μm (C and D), 10 μm (E). (F) Immunofluorescence staining of the ARF-DTR lung. Sections were costained with surfactant-associated protein C (SP-C) or the ER-TR7 fibroblast marker together with luciferase. Sections were counterstained with DAPI. Original magnification, ×100; scale bar: 10 μm. (G) The luciferase-positive population in SP-C– or ER-TR7–stained cells was counted. At least 100 SP-C– or ER-TR-7–positive cells were analyzed in each section. Values represent the mean ± SEM of 3 independent experiments. N.D., not detected. (H) Gating strategy for CD31–; CD45–; EpCAM+ epithelial cells and CD31–; CD45–; Sca-1+ mesenchymal cells. Lung cells from 7 ARF-DTR mice were pooled before sorting. (I) Luciferase activity was determined in gated epithelial and mesenchymal cells shown in (H). Luciferase activity was normalized to cell numbers in each sample. A representative result of 2 independent experiments is shown. Values represent the mean ± SD of triplicate samples. *P < 0.05, unpaired Student’s t test.