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. 2012 Jun;122(6):2032-45.
doi: 10.1172/JCI60132. Epub 2012 May 1.

SIRT1 protects against emphysema via FOXO3-mediated reduction of premature senescence in mice

Hongwei Yao  1 , Sangwoon Chung, Jae-woong Hwang, Saravanan Rajendrasozhan, Isaac K Sundar, David A Dean, Michael W McBurney, Leonard Guarente, Wei Gu, Mikko Rönty, Vuokko L Kinnula, Irfan Rahman
Affiliations

SIRT1 protects against emphysema via FOXO3-mediated reduction of premature senescence in mice

Hongwei Yao et al. J Clin Invest. 2012 Jun.

Abstract

Chronic obstructive pulmonary disease/emphysema (COPD/emphysema) is characterized by chronic inflammation and premature lung aging. Anti-aging sirtuin 1 (SIRT1), a NAD+-dependent protein/histone deacetylase, is reduced in lungs of patients with COPD. However, the molecular signals underlying the premature aging in lungs, and whether SIRT1 protects against cellular senescence and various pathophysiological alterations in emphysema, remain unknown. Here, we showed increased cellular senescence in lungs of COPD patients. SIRT1 activation by both genetic overexpression and a selective pharmacological activator, SRT1720, attenuated stress-induced premature cellular senescence and protected against emphysema induced by cigarette smoke and elastase in mice. Ablation of Sirt1 in airway epithelium, but not in myeloid cells, aggravated airspace enlargement, impaired lung function, and reduced exercise tolerance. These effects were due to the ability of SIRT1 to deacetylate the FOXO3 transcription factor, since Foxo3 deficiency diminished the protective effect of SRT1720 on cellular senescence and emphysematous changes. Inhibition of lung inflammation by an NF-κB/IKK2 inhibitor did not have any beneficial effect on emphysema. Thus, SIRT1 protects against emphysema through FOXO3-mediated reduction of cellular senescence, independently of inflammation. Activation of SIRT1 may be an attractive therapeutic strategy in COPD/emphysema.

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Figures

Figure 1
Figure 1. SIRT1 protects against airspace enlargement and increased lung compliance in emphysematous mice.
(A–D) Sirt1+/– mice were susceptible to developing airspace enlargement (A and B), whereas overexpression of Sirt1 (i.e., Sirt1 Tg mice) attenuated the increased Lm of airspace in response to 6 months of CS exposure (C and D). (E and F) Lung compliance (C) was increased in Sirt1+/– mice compared with WT littermates in response to 6 months of CS exposure (E), which was attenuated by Sirt1 overexpression (F). (G and H) Sirt1 deficiency increased the airspace enlargement induced by intratracheal injection of elastase (Ela). Sal, saline. (I) Lung compliance was increased in Sirt1+/– mice compared with WT littermates after elastase exposure. H&E-stained images are representative of experiments from 3 separate mice. Original magnification, ×100. Scale bars: 100 μm. n = 3–4 per group. *P < 0.05, **P < 0.01, §P < 0.001 versus air or saline; #P < 0.05, ##P < 0.01 versus WT.
Figure 2
Figure 2. SRT1720 exhibits protective and therapeutic effects on elastase-induced airspace enlargement and increase in lung compliance.
(A and B) Administration of SRT1720 (SRT; 50–100 mg/kg) by oral gavage during emphysema development significantly prevented elastase-induced increase in Lm of airspace (A) and lung compliance (B) in 129/SvJ WT mice. (C and D) Administration of SRT1720 (100 mg/kg) during emphysema development did not alter the airspace enlargement (C) or increased lung compliance (D) in Sirt1+/– mice. (E and F) Oral administration of SRT1720 (100 mg/kg) after the development of emphysema attenuated airspace enlargement (E) and increased lung compliance (F) in WT, but not Sirt1+/–, mice. Veh, vehicle. n = 3–4 per group. §P < 0.001 versus saline; #P < 0.05, ##P < 0.01, ###P < 0.001 versus vehicle; ††P < 0.01, †††P < 0.001 versus WT.
Figure 3
Figure 3. Elastase-induced airspace enlargement and lung function decline are further aggravated in Epi-Sirt1–/–, but not in Mac-Sirt1–/–, mice.
(A and B) Elastase-induced airspace enlargement was increased in Epi-Sirt1–/– mice compared with WT littermates. (C) Deficiency of Sirt1 in airway epithelium led to an increase in lung compliance in response to elastase exposure. (D) RL was further reduced in Epi-Sirt1–/– mice exposed to elastase. (E and F) No significant difference in Lm of airspace was seen between Mac-Sirt1–/– and WT mice after elastase intratracheal injection. (G and H) The myeloid cell–specific deficiency of Sirt1 (in Mac-Sirt1–/– mice) did not affect lung compliance (G) or RL (H) in response to elastase exposure. Original magnification, ×100. Scale bars: 100 μm. n = 3–4 per group. **P < 0.01, §P < 0.001 versus saline; #P < 0.05, ##P < 0.01 versus WT.
Figure 4
Figure 4. Foxo3 deficiency diminishes the protection of SRT1720 against elastase-induced emphysema.
(A and B) SRT1720-mediated protection against elastase-induced airspace enlargement was diminished in Foxo3–/– mice. (C and D) No effect of SRT1720 on lung compliance (C) or RL (D) was observed in Foxo3–/– mice exposed to elastase. (E and F) Treadmill running time (E) and arterial oxygen saturation (F) were decreased in Foxo3–/– mice in response to elastase intratracheal injection, which was not altered by SRT1720. H&E-stained images are representative of experiments from 3 separate mice. Original magnification, ×100. Scale bar: 100 μm. n = 3–4 per group. **P < 0.01, §P < 0.001 versus saline; ##P < 0.01 versus WT; †P < 0.05, ††P < 0.01 versus vehicle.
Figure 5
Figure 5. SIRT1 protects against CS-induced increase in p21, p16, and p53.
Lung levels of p21, p16, and p53 were increased in Sirt1+/– mice versus WT littermates exposed to CS for 6 months, which was attenuated by Sirt1 overexpression. No significant change was observed in p27 level among these mice. Blots are representative of experiments from 3 separate mice. Band density is expressed as fold change relative to corresponding β-actin. n = 3–4 per group. *P < 0.05, **P < 0.01, ***P < 0.001 versus air; ###P < 0.001 versus WT.
Figure 6
Figure 6. SIRT1 protects against CS- or elastase-induced increase in lung SA–β-gal activity.
(A) CS exposure for 3 days increased lung SA–β-gal activity (arrows). (B) Whereas SA–β-gal activity was increased by Sirt1 deficiency when exposed to CS for 6 months, this was not observed in lungs of Sirt1 Tg mice. (C) SRT1720 administration prior to emphysema development attenuated SA–β-gal activity in lungs of WT, but not Sirt1+/–, mice in response to elastase exposure. (D) SRT1720 administration after emphysema development for 2 weeks ameliorated elastase-induced increase in lung SA–β-gal activity (arrows). (E and F). SA–β-gal activity was increased in lungs of Epi-Sirt1–/–, but not Mac-Sirt1–/–, mice compared with their WT littermates after elastase injection. Original magnification, ×200; ×400 (inset). Scale bars: 100 μm. SA–β-gal activity is expressed as 4-MU fluorescence normalized to protein content (see Methods). n = 3–4 per group. ‡P < 0.05 versus air; *P < 0.05, **P < 0.01, §P < 0.001 versus saline; #P < 0.05 versus WT; ††P < 0.01 versus vehicle.
Figure 7
Figure 7. CS-induced lung cellular senescence is increased in Foxo3–/– mice.
(A) Lung level of p21 was increased in Foxo3–/– mice versus WT littermates exposed to CS for 4 months. (B) Foxo3 deficiency increased the levels of p16 and p27, but not p53, in lungs of mice exposed to CS for 4 months. (C) SA–β-gal activity was increased in lungs of Foxo3–/– mice compared with WT littermates after 4 months of CS exposure. Blots are representative of experiments from 3 separate mice. Band density is expressed as fold change relative to corresponding β-actin. SA–β-gal activity is expressed as 4-MU fluorescence normalized to protein content (see Methods). n = 3 per group. *P < 0.05, **P < 0.01, ***P < 0.001 versus air; #P < 0.05, ##P < 0.01, ###P < 0.001 versus WT.
Figure 8
Figure 8. SRT1720 protection against lung senescence is diminished in Foxo3–/– mice exposed to elastase.
(A) SRT1720 administration before emphysema development reduced elastase-induced increase in the level of p21 in WT mouse lung, which was diminished in Foxo3–/– mice. (B) Elastase-induced increase in SA–β-gal activity was attenuated by SRT1720 in WT mice, which was diminished in Foxo3–/– mice. (C) Attenuation in levels of p16 and p53, but not p27, in lungs by SRT1720 was diminished in Foxo3–/– mice exposed to elastase. Blots are representative of experiments from 3 separate mice. Band density is expressed as fold change relative to corresponding β-actin. SA–β-gal activity is expressed as 4-MU fluorescence normalized to protein content (see Methods). n = 3 per group. **P < 0.01, §P < 0.001 versus saline; #P < 0.05, ##P < 0.01 versus WT; †P < 0.05, ††P < 0.01 versus vehicle.
Figure 9
Figure 9. p21 deficiency ameliorates CS-induced airspace enlargement and lung function decline and protects against both CS- and sirtinol-induced increase in SA–β-gal activity.
(A and B) CS exposure for 6 months led to airspace enlargement in WT mice, which was attenuated in p21–/– mice. (C) p21 deficiency protected against increased lung compliance induced by 6 months of CS exposure. (D) No alteration of RL was observed in either WT or p21–/– mice exposed to CS for 6 months. (E) Genetic ablation of p21 attenuated 6 months of CS-mediated increase in SA–β-gal activity in mouse lung. (F) Sirtinol (Sir) treatment further increased SA–β-gal activity in lungs of WT mice, but not P21–/– mice, in response to 3 days of CS exposure. H&E-stained images are representative of experiments from 3 separate mice. Original magnification, ×100. Scale bar: 100 μm. SA–β-gal activity is expressed as 4-MU fluorescence normalized to protein content (see Methods). n = 3–4 per group. *P < 0.05, **P < 0.01, §P < 0.001 versus air; #P < 0.05, ##P < 0.01 versus WT; †P < 0.05 versus vehicle.
Figure 10
Figure 10. The IKK2 inhibitor PHA-408 does not influence elastase-induced emphysema or increase in SA–β-gal activity in lungs of either Sirt1+/– or WT mice.
(A and B) Treatment with PHA-408 (PHA) did not alter elastase-induced increase in Lm of airspace in either Sirt1+/– mice or WT littermates. (C and D) Lung compliance (C) or RL (D) was not affected by PHA-408 in either WT or Sirt1+/– mice exposed to elastase. (E and F) Decreased arterial oxygen saturation (E) or running time (F) induced by elastase was not affected by PHA-408 treatment. (G) Elastase-induced increase in SA–β-gal activity was not altered by PHA-408. H&E-stained images are representative of experiments from 3 separate mice. Original magnification, ×100. Scale bar: 100 μm. SA–β-gal activity is expressed as 4-MU fluorescence normalized to protein content (see Methods). n = 3–4 per group. *P < 0.05, **P < 0.01, §P < 0.001 versus saline; #P < 0.05, ##P < 0.01 versus vehicle.
Figure 11
Figure 11. Role of the SIRT1/FOXO3 pathway in protecting against emphysema.
Lung level of SIRT1 is reduced in response to CS or oxidative/carbonyl stress, which leads to acetylation and degradation of FOXO3 and culminates in stress-induced premature senescence. The senescence of lung cells is a contributing factor to airspace enlargement and emphysema. NF-κB is also acetylated and activated in response to CS exposure as a result of SIRT1 reduction. However, inhibition of NF-κB–dependent lung inflammation does not prevent the progression of airspace enlargement. SIRT1 activation by overexpression and pharmacological means protects the lungs against cellular senescence and emphysematous changes.
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