We and others have previously shown that the level of SIRT1 is substantially decreased in lungs of patients with COPD/emphysema as well as in lungs of rodents exposed to CS (17
). However, the role of endogenous SIRT1 in the development of emphysema remains elusive. We therefore studied the role of SIRT1 in the pathogenesis of emphysema in mice using various genetic and pharmacological approaches. Our findings indicate that SIRT1 protected against CS- and elastase-induced airspace enlargement, decline in lung function, impaired exercise endurance, and decreased arterial oxygen saturation, which are the characteristic features of COPD/emphysema. Furthermore, Sirt1
deletion in airway epithelium, but not in myeloid cells, aggravated airspace enlargement and lung function decline induced by elastase. Collectively, these observations suggest that SIRT1 exhibits a cell-specific protective role in emphysema. SIRT1 level and activity were reduced in an age-dependent manner in rodent lungs, as shown in this study and supported by others (39
). Interestingly, the Sirt1
-deficient mice developed spontaneous airspace enlargement only after 1 year of age, although a significant reduction in SIRT1 occurred at 6–8 months of age in the lungs of these mice. Additionally, Sirt1
-deficient mice at 6 months of age developed emphysema after CS exposure for 4 months, whereas 6 months of CS exposure was required to develop emphysema in WT mice. This suggests that SIRT1 reduction in early life is not enough to cause lung injury, but increases the susceptibility to develop stress-induced emphysema.
In agreement with the previous studies (40
), chronic CS exposure did not alter RL
, which was significantly decreased in elastase-exposed mice, suggesting that the reduction of RL
occurs only in the setting of more severe emphysema. This was further corroborated by the findings in Sirt1
-deficient mice, which exhibited lower RL
and enhanced airspace enlargement than did WT littermates in response to both chronic CS and elastase exposures. However, the Rn was not altered by either CS or elastase exposure, or loss or gain of function of SIRT1. These results suggest that peripheral airway resistance is lower in the condition of severe emphysema, which may be due to increased destruction of small airways. These findings in the mouse model of emphysema are in contrast to the increased RL
seen in human COPD (42
). This may be attributed to the anatomical features of mouse lung, such as the relatively large airway size and lack of submucosal glands, which might not cause the narrowing and obstruction of conducting airways (43
). We noted that the Lm value was about 50–60 μm in air- and saline-exposed WT control mice, which is consistent with previous findings (44
). However, some studies have shown that the Lm of airspace is about 25–35 μm in WT control mice (47
). The discrepancy in Lm of airspace among these studies may be associated with the differences in CS doses, pattern/composition of smoke delivered by different CS generating systems, mouse strains, and techniques used for measuring Lm. Nonetheless, 6 months of CS exposure and elastase intratracheal injection increased the Lm of airspace by approximately 19% and 38%, respectively. This was corroborated by the previous findings showing 15%–20% increase of Lm by chronic CS exposure, and 25%–45% increase by elastase administration (45
SIRT1 deacetylates FOXO3 via direct protein-protein interaction, thereby tipping the balance to cellular survival in response to oxidative/carbonyl stress (11
). Our previous study showed an increase in both FOXO3 degradation and acetylation in lungs of COPD patients and mouse lung exposed to CS (51
). This was due to the reduction of SIRT1 level and its interaction with FOXO3 in response to CS exposure. However, it remains to be seen which residues of FOXO3 are acetylated by CS and regulated by SIRT1. Furthermore, the study is required to determine whether the increased acetylation marks FOXO3 for its degradation, as well as alters its transactivation on target genes (e.g., prosenescent versus antioxidant genes). Nevertheless, we have shown that Foxo3
deficiency increases the susceptibility of mice to develop emphysema (51
). Interestingly, the protective effect of a selective pharmacological SIRT1 activator, SRT1720, against emphysema was diminished in Foxo3
KO mice. These findings suggest that the beneficial effect of SIRT1 on emphysema requires FOXO3.
Accumulating evidence supports the notion that COPD is a disease of accelerated and premature aging, as enhanced oxidative stress and cellular senescence occur in lungs and systemic circulation of patients with this disease (3
). Therefore, we proposed that an age-dependent cellular senescence would be a target for the protection of SIRT1 against emphysema in mice. CS exposure significantly induced premature senescence in mouse lung, which was attenuated by SIRT1 overexpression and by its activator, SRT1720. This is corroborated by the finding that SIRT1 protects against telomere shortening and erosion (a biological maker of replicative senescence) (53
). However, the role of SIRT1 in CS-induced replicative senescence is unclear, although telomere length is a determinant of emphysema susceptibility (54
). Furthermore, the lung levels of p16, p21, and p27, as well as SA–β-gal activity, were further increased in Foxo3
-deficient mice with emphysema, which was supported by a prior study showing the protection of FOXO3 against cellular senescence (14
). Strikingly, Foxo3
deficiency diminished the effect of SRT1720 in attenuating the levels of p21 and p16 as well as SA–β-gal activity in emphysematous lungs, indicative of the requirement of FOXO3 for SIRT1’s protection against SIPS. Importantly, deletion of p21 significantly ameliorated CS-induced airspace enlargement and lung function decline. Both CS and sirtinol induced an increase in SA–β-gal activity in mouse lung, which was significantly attenuated by p21
deficiency. Hence, SIRT1 activation downregulated SIPS through FOXO3/p21 pathway, thereby protecting against emphysema. In addition to FOXO3, recent studies have demonstrated the involvement of other developmental and senescence-related genes, such as Wnt/β-catenin, Notch, Klotho, senescence marker protein-30, and Werner syndrome protein, in the development of emphysema (55
). However, it remains to be seen whether SIRT1 targets these genes in response to CS exposure.
SIRT1 has been shown to upregulate FOXO3-dependent antioxidant genes (i.e., catalase and MnSOD) and to protect against oxidative stress–induced cellular apoptosis (61
). Moreover, FOXO3 forms a complex network along with p53 in regulating cellular responses to oxidative stress, such as senescence, proliferation, and apoptosis (11
). This suggests the involvement of SIRT1/FOXO3/p53-dependent signaling in regulating cellular senescence. Both oxidative stress and apoptosis play an important role in the development of COPD/emphysema (44
). Hence, it is likely that SIRT1 augmentation alleviates emphysema via downregulating oxidative stress–mediated cellular senescence and apoptosis.
Chronic CS exposure reduced the level of SIRT1 in BAL cells (mainly composed of macrophages) and lung epithelial cells in mice. This is consistent with our previous studies showing SIRT1 reduction in monocytes/macrophages, lung epithelial cells, endothelial cells, and fibroblasts treated with CS extract in vitro (18
). Interestingly, SA–β-gal activity in lungs was increased in mice with SIRT1 deficiency in Clara cells, but not in myeloid cells, compared with corresponding WT littermates in response to elastase administration. Furthermore, the SA–β-gal–positive cells were mainly localized in the airway epithelium of emphysematous mice and COPD patients. These results indicate the importance of SIRT1 reduction associated with senescence in Clara cells (the progenitor cells of the peripheral airway epithelium) in the development of emphysema. This is in agreement with increased number of senescent Clara cells in lungs of patients with COPD compared with nonsmokers (52
). Nevertheless, the possibility that SIRT1 regulates senescence in fibroblasts and endothelial cells cannot be excluded. In addition, the SIRT1/FOXO3 axis may be involved in the regulation of lymphocyte senescence, thereby preventing the recognition of self-antigens particularly in mice with emphysema, since SIRT1 attenuated autoimmunity reaction by inhibiting T cell activation (9
Inflammation and cellular senescence are intertwined in the process of accelerated or premature lung aging (inflammaging) (71
). The percentage of proinflammatory senescent type II cells expressing both p16 and phosphorylated NF-κB (i.e., senescence-associated secretory phenotype [SASP]) has been shown to be augmented in lungs of COPD patients compared with smokers and nonsmokers (33
). Senescent cells are prone to generate proinflammatory mediators, which may reinforce the senescence growth arrest or mobilize innate immune cells to clear senescent/senesced cells (72
). Consistent with this, both SIRT1 and genetic disruption of the prosenescent gene p21
attenuated CS-induced lung inflammation, which was associated with reduced NF-κB activation (34
). Interestingly, the inhibition of lung inflammation using the selective NF-κB/IKK2 inhibitor PHA-408 did not affect cellular senescence or emphysematous destruction. This observation suggests that NF-κB–dependent lung inflammation does not contribute to lung dysfunction or that it is just one of the consequences of cellular senescence.
It has previously been shown that SIRT1 negatively regulates MMP-9 by lowering NF-κB activation (17
). We found that the level and activity of MMP-9 were further increased in lungs of Sirt1
-deficient mice, which were attenuated by Sirt1
overexpression in response to CS exposure (H. Yao and I. Rahman, unpublished observations). Furthermore, mice overexpressing MMP-9 develop lung emphysematous phenotype, whereas MMP-9–deficient mice are protected from IL-13–induced airspace enlargement (75
). These findings suggest the possible involvement of MMPs in SIRT1-mediated regulation of emphysema via an unknown mechanism.
In conclusion, SIRT1 protected against emphysema through a FOXO3-dependent antisenescent mechanism. Furthermore, the inhibition of NF-κB–dependent inflammation with PHA-408 did not exhibit any protective effect in elastase-induced airspace enlargement or decline in lung function. Therefore, the antisenescent, but not antiinflammatory, property contributes to the protection of SIRT1 against emphysema (Figure ). These findings highlight the mechanism of SIPS in the pathogenesis of COPD/emphysema. They also provide the rationale for a key and specific therapeutic target via pharmacological activation of SIRT1 in ameliorating/halting the progression of this diverse and complex debilitating disease (71
). Hence, the activation of SIRT1 may prove a therapeutic intervention to prevent premature lung senescence/aging in COPD.
Role of the SIRT1/FOXO3 pathway in protecting against emphysema.