In order to investigate the role of p19ARF in tissue aging, we established a transgenic mouse (ARF–diphtheria toxin receptor [ARF-DTR]) in which the DTR (human HB-EGF I117V/L148V; ref. 14) and luciferase were expressed from the CDKN2A locus (Figure 1A). Embryonic fibroblasts prepared from these mice (MEFs) expressed p19ARF, p16INK4a, p21, and luciferase when senescence was induced by either serial passages or oncogenic Ras (Figure 1, B and C). The expression of senescence markers (p19ARF and p16INK4a) as well as luciferase was diminished when senescent ARF-DTR MEFs were treated with diphtheria toxin (DT) (Figure 1, D–G). An in vivo imaging analysis revealed that luciferase activity was barely detectable in young (2-month-old) female mice but became apparent in multiple tissues by the age of 12 months (Figure 2A). Similar results were obtained in male mice, with the exception of the testis (data not shown); luciferase was detectable in the testis irrespective of the age of animals, which presumably reflected the lifelong expression of p19ARF in mouse spermatogonia (16). The luminescence signals observed in old female mice were attributed to those in the lungs and adipose tissues (Figure 2, C and D), which is consistent with the endogenous expression of ARF in the lungs and adipose tissues at this age (Figure 2B). INK4a mRNA expression was also increased, as previously reported (8), confirming the accumulation of senescent cells in these tissues. However, expression of ARF is increased in many tissues during aging (8), suggesting that the expression of the transgene in ARF-DTR mice does not mimic the expression pattern of endogenous ARF in other tissues.

Figure 1 Establishment of ARF-DTR transgenic mice. (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.

Figure 2 A luciferase signal was detected in 12-month-old ARF-DTR transgenic mice. (A) D-luciferin (150 mg/kg body weight) was injected intraperitoneally into 2- or 12-month-old female ARF–diphtheria toxin receptor (ARF-DTR) or 12-month-old female wild-type mice. Ten minutes after the injection of luciferin, an in vivo imaging analysis was performed in order to detect luminescence with a 3-minute exposure. Mice were shaved before the injection. (B) Total RNA was extracted from wild-type mouse lung and perigonadal adipose tissues at the indicated ages. ARF and INK4a mRNA levels were analyzed by real-time PCR and normalized to that of 18S rRNA. Values represent mean ± SEM of at least 3 independent experiments. (C) Luciferase activity was monitored in 12-month-old female ARF-DTR mice before (left) and 2 days (right) after an intraperitoneal injection of diphtheria toxin (DT). Signals in the red circle indicate lung tissue luciferase activity. (D) Luciferin was injected intraperitoneally into 12-month-old female ARF-DTR mice treated or untreated with DT for 3 days. Ten minutes later, mice were sacrificed and subjected to laparotomy, and in vivo luciferase activity was analyzed. A light image (left) and superimposed image (right) are shown. Lu, lung; Ad, adipose tissue. Representative images of 3 independent experiments are shown.

The intraperitoneal administration of DT eliminated the luminescence signal from the lungs within 48 hours (Figure 2, C and D), and this effect lasted for at least 2 weeks after drug administration (Supplemental Figure 1; supplemental material available online with this article; doi:10.1172/jci.insight.87732DS1). In contrast, the DT treatment failed to reproducibly decrease luciferase activity in adipose tissue for an unknown reason. Based on the above results, we analyzed the role of p19ARF-expressing cells in lung function using female ARF-DTR mice.

The aging-associated decline in lung function is characterized by an increase in tissue compliance due to the progressive loss of tissue elasticity, which results in the incomplete contraction of lung tissue during the expiration phase (17–19). Luciferase was not detected in bronchoalveolar lavage fluid cells but was detected in the fraction mainly containing fibroblasts (Figure 3, A and B). Immunohistochemistry revealed that endogenous p19ARF colocalized with luciferase and the senescence marker γH2AX in 12-month-old lung tissue (Figure 3, D and E). Luciferase was observed in cells expressing fibroblast markers (vimentin and ER-TR7) but not in those expressing the alveolar epithelial marker, SP-C (Figure 3, C, F, and G). Furthermore, when lung cells were sorted according to epithelial (CD31–; CD45–; EpCAM+) and mesenchymal

Figure 3 Luciferase was expressed in alveolar fibroblasts in old ARF-DTR mice. (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.

(CD31–; CD45–; Sca-1+) markers, luciferase was detected in the mesenchymal population, suggesting that p19ARF-expressing cells in the adult mouse lung are alveolar fibroblasts (Figure 3, H and I). Taken together, these results strongly indicate that cellular senescence is induced in the alveolar fibroblasts of the 12-month-old lung parenchyma.

Twelve-month-old mice were treated with PBS or DT (Figure 4A). Lung luciferase activity in 12-month-old mice decreased to a similar level as that in young mice after the DT treatment (Figure 4B). No significant changes were observed in body weights after the DT treatment (Supplemental Figure 2). The DT treatment reduced ARF, INK4a, and p21 levels in the lungs of ARF-DTR mice, but not in those of wild-type mice, indicating that p19ARF-expressing senescent cells were successfully removed from lung tissue of ARF-DTR mice (Figure 4, C and D).

Figure 4 Downregulation of senescence genes by eliminating p19ARF-expressing cells. (A) Experimental design. Twelve-month-old female wild-type or ARF–diphtheria toxin receptor (ARF-DTR) mice were intraperitoneally injected with diphtheria toxin (DT) or PBS twice with a 2-week interval. Two weeks after the second DT injection, mice were sacrificed and their lungs were analyzed. (B) Luciferase signals in the lung area were measured in ARF-DTR mice using Living Image in vivo imaging software. Values represent the mean ± SEM of independent experiments. (C and D) Total RNA was extracted from the lungs of 2-month-old and 12-month-old female ARF-DTR mice treated with DT or not treated for 4 weeks (C) or 12-month-old wild-type mice treated or not treated with DT for 4 weeks (D). The expression of ARF, INK4a, and p21 was analyzed by real-time PCR. mRNA levels were normalized to GAPDH in each mouse. Values represent the mean ± SEM of independent experiments. Data were analyzed 1-way ANOVA followed by post-hoc Tukey-Kramer multiple comparison test. *P < 0.05, **P < 0.01, ***P < 0.001.

We then performed pulmonary function tests on these mice. As previously reported (20, 21), static lung compliance (Cst) was significantly higher in older animals than in young animals (Figure 5, A and B). The DT treatment resulted in the marked recovery of lung elasticity (decrease in Cst) in ARF-DTR mice but not in wild-type mice. Similarly, the DT treatment reversed aging-associated changes in dynamic compliance, dynamic resistance, tissue elastance, and tissue damping in ARF-DTR mice but not in wild-type mice (Figure 5, C–F). These results clearly indicated that the p19ARF-expressing cells that accumulated in 12-month-old lung tissue had deleterious effects on pulmonary function and that aging-associated declines in pulmonary function were ameliorated by the elimination of these p19ARF-expressing cells.

Figure 5 Restoration of lung function by eliminating p19ARF-expressing cells. (A) Pressure-volume curves of ARF–diphtheria toxin receptor (ARF-DTR) and wild-type mouse lungs treated with diphtheria toxin (DT) or PBS, as depicted in Figure 4A. Mice in the indicated groups were euthanized and connected to the FlexiVent system through their tracheae. Mice were subjected to laparotomy, and diaphragms were removed prior to the assay. (B) Static lung compliance (Cst) was calculated from the slope of the pressure-volume loop. Cst reflects the static elastic recoil pressure of the lungs at a given lung volume. (C–F) Dynamic lung compliance (Crs) (C), dynamic lung resistance (Rrs) (D), tissue elastance (H) (E), and tissue damping (G) (F) are shown. Dynamic lung compliance captures the ease with which the lungs may be extended, and dynamic lung elastance captures the elastic rigidity or stiffness of the lungs. Tissue elastance and tissue damping reflect energy conservation and dissipation in lung alveoli, respectively. Values represent mean ± SEM of independent experiments. Data were analyzed 1-way ANOVA followed by post-hoc Tukey-Kramer multiple comparison test. *P < 0.05, **P < 0.01, ***P < 0.001.

We also compared lung morphologies among these mice. An increase in alveolar size is associated with a decline in lung function during aging (18, 19, 22, 23). Alveolar mean linear intercepts (the average distance between alveolar walls that reflects the size of the alveolar space; ref. 24) were increased in 12-month-old ARF-DTR mice (Figure 6, A and B). The DT treatment reversed this phenotype but did not affect alveolar size in wild-type mice. Mean alveolar septal wall thickness was lower in 12-month-old mice than in 2-month-old mice (Figure 6C), as previously reported (25). Furthermore, the ablation of p19ARF-expressing cells fully restored the alveolar wall in ARF-DTR mice but not in wild-type mice. Increases in lung compliance in aged animals have been attributed to a decline in the number of elastic fibers, the major component of which is elastin (19, 22). Therefore, we performed a quantitative analysis on elastin in immunostained sections (Figure 6D). The amount of elastin in older animals was approximately half that in young animals (Figure 6E) and recovered in DT-treated ARF-DTR mice but not in wild-type mice.

Figure 6 Effects of p19ARF-expressing cell elimination on lung morphology. (A) Mice were treated with diphtheria toxin (DT) or PBS, as depicted in Figure 4A. Lung tissues were fixed with Bouin solution at 25 cmH 2 O and subjected to Resorcin-Fuchsin staining, followed by counterstaining with hematoxylin-eosin. Original magnification, ×120 (using digital zoom); scale bar: 20 μm. (B and C) Alveolar mean linear intercepts (B) and septal wall thickness (C) were measured. More than 400 alveoli and 150 septal walls per mouse were counted. (D) Images of the elastin immunostaining of lung sections. Lung sections were immunostained with anti-elastin and visualized using a HRP-conjugated secondary antibody. Immunostaining in each sample was performed under identical conditions. Original magnification, ×40. (E) Captured elastin immunostaining images were analyzed by ImageJ for the quantitation of elastin signals per field. Values represent the mean ± SEM of independent experiments. Data were analyzed 1-way ANOVA followed by post-hoc Tukey-Kramer multiple comparison test. *P < 0.05, **P < 0.01, ***P < 0.001.

We also examined the effects of ARF-expressing cell elimination on even older animals. Tumor-free female ARF-DTR or wild-type mice between 20 and 22 months old were treated with PBS or DT for 1 month (Figure 7A). In vivo imaging analysis confirmed that luciferase signal disappeared from lung tissue (Figure 7B). Pulmonary function tests revealed that tissue compliance in older mice was similar to that in 12-month-old mice (Figure 7, C, D, and F). The DT treatment reduced tissue compliance in older animals; however, this effect was less than that observed in 12-month-old mice. Similarly, the DT treatment reversed aging-associated decreases in dynamic resistance and tissue damping (Figure 7, E and G). Alveolar size, which was larger than that in 12-month-old mice, was slightly restored in DT-treated animals (Figure 7, H and I). Collectively, these results indicated that p19ARF-expressing cells provoked the loss of elastic fibers in lung tissue and were also responsible for the increase in lung compliance in aged animals.

Figure 7 Effects of the DT treatment on older mice. (A) Experimental design. Twenty- to twenty-two-month-old female ARF–diphtheria toxin receptor (ARF-DTR) or wild-type mice were intraperitoneally injected with diphtheria toxin (DT) or PBS twice with a 2-week interval. Two weeks after the second DT injection, mice were sacrificed and their lungs were analyzed. (B) Luciferase activity was monitored in ARF-DTR mice before (Pre-DT) and after (Post-DT) DT treatment. (C) Pressure-volume curves of mouse lungs treated with DT or PBS. (D–G) Static lung compliance (Cst), dynamic resistance (Rrs), dynamic compliance (Crs), and tissue damping (G). (H) Images of the lungs of PBS-and DT-treated mice. Sections were subjected to Resorcin-Fuchsin staining followed by counterstaining with hematoxylin-eosin staining. Original magnification, ×120 (using digital zoom); scale bar: 10 μm. (I) Alveolar mean linear intercepts of PBS-and DT-treated mice were shown. Values represent mean ± SEM of independent experiments. *P < 0.05, unpaired Student’s t test.

Senescent cells express cell cycle–regulating genes as well as a series of genes that function non–cell autonomously, such as cytokines, thereby affecting the behaviors of their surrounding “nonsenescent” cells (26, 27). In order to gain an insight into the relationship between p19ARF-expressing cells and aging-associated phenotypes in lungs, we examined the expression of senescence-associated genes in these animals (Figure 8A). Among the MMPs examined, MMP-10 and -12 were significantly increased in older mice and were completely abolished or diminished in DT-treated ARF-DTR mice but not in wild-type animals (Supplemental Figure 3). These MMPs have been shown to exhibit elastase activities (28, 29) and are, thus, good candidates for the reduced level of elastin observed in the lungs of aged animals. MMP-12 knockout mice were found to resist the degradation of elastin when emphysema was induced by cigarette smoking (30). MMP-10 protein was also downregulated in DT-treated ARF-DTR lungs but not to the same extent as their mRNA levels (Supplemental Figure 4). In order to ascertain whether increases in MMP were responsible for the reduced elastin level, MEFs were infected with retroviruses encoding shRNA against MMP-10. Cells were then subjected to ionizing radiation and cultured under 3% oxygen to induce senescence-associated genes in MEFs (31). MMP-10 level was decreased to approximately half by shRNA (Figure 8B). Elastin levels were reduced to approximately half that of untreated cells cultured under 20% oxygen (data not shown) but recovered when MMP activities were inhibited either by shRNA or a chemical inhibitor (Figure 8C). Taken together, these results suggest that the loss of elastin in senescent cells was, at least partly, attributed to increased MMP.

Figure 8 Changes in senescence-associated gene expression in DT-treated lungs. (A) Total RNA was extracted from 2- and 12-month-old female ARF–diphtheria toxin receptor (ARF-DTR) mouse lung tissues. Twelve-month-old mice were treated with PBS or diphtheria toxin (DT), as depicted in Figure 4A. The expression of the indicated genes was analyzed by real-time PCR. mRNA levels were normalized to GAPDH mRNA in each sample. Values represent the mean ± SEM. (B) Wild-type MEFs were infected with retroviruses encoding shRNA against MMP-10. Two-independent shRNA were used to knockdown MMP-10. Scrambled short hairpin (sh-SCR) was used as a control in each group. Infected cells were selected with puromycin for 3 days, exposed to ionizing radiation (10 Gy), and cultured under 3% oxygen conditions for 10 days. The expression of MMP-10 was analyzed by immunoblotting. Lamin A/C was used as a loading control. (C) shRNA-infected senescent MEFs were analyzed for the amount of elastin. In the chemical inhibition of MMPs, senescent MEFs were cultured in the presence of an MMP inhibitor [N-isobutyl-N-(4-methoxyphenylsulfonyl)-glycylhydroxamic acid {NNGH}]. Media were replaced every 3 days. The amount of elastin was measured and then normalized to the total protein content in each sample. Values represent the mean ± SD of triplicate samples. Data are representative of 2 independent experiments. Data were analyzed 1-way ANOVA followed by post-hoc Tukey-Kramer multiple comparison test (for comparison of 3 groups) or unpaired Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.001.

We also compared gene expression profiles among 2-month-old and 12-month-old (with or without DT) ARF-DTR lungs. Genes differentially expressed (more than 2-fold) between 2-month-old and 12-month-old lungs were identified (Supplemental Table 1, a full list of genes is available from the GEO database, accession GSE64754; http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE64754). Changes in the expression of these genes among young and old DT-treated animals were then analyzed. In order to simplify the results obtained, genes with expression levels that changed more than 2-fold in wild-type animals after the DT treatment were subtracted from the list because they exhibited “nonspecific” responses to DT. A pathway analysis revealed that genes involved in cytokine and chemokine signaling are differentially expressed in 12-month-old lungs, which may reflect the secretory phenotype of senescent cells (26, 27) (Table 1). Of the 305 genes that were upregulated in old lungs, the expression of 170 (55.7%) genes was decreased following the DT treatment (Figure 9 and Supplemental Table 1). Conversely, the DT treatment upregulated the expression of 82 (62.1%) of the 132 downregulated genes in old lungs. Thus, the elimination of p19ARF-expressing cells led to the “rejuvenation” of gene expression profiles in whole lung tissue; however, approximately 40% of genes with altered expression levels in 12-month-old animals remained intact under these conditions, suggesting that p19ARF contributed to only a certain aspect of the aging-associated phenotypes of the lung or that some aging-associated changes are irreversible.

Figure 9 Effect of p19ARF-expressing cell elimination on aging-associated gene expression in lung tissue. Twelve-month-old mice were treated with PBS or diphtheria toxin (DT) for 4 weeks, as depicted in Figure 4A, before the analysis. Four mice were independently analyzed in each group, and average values were used to draw heatmaps. Genes that nonspecifically responded to DT were subtracted from the list. The genes used to draw the heatmap are listed in Supplemental Table 1. A full list of genes is available from the GEO database (accession GSE64754). *P < 0.05, **P < 0.01, ***P < 0.001