Recent focus on COPD research is to understand the factors or molecular mechanisms involved in underlying pathogenesis of lung inflammation in smokers who are susceptible to COPD. We have previously shown that proinflammatory cytokine release was increased in a human monocytic–macrophage cell line (MonoMac6) in vitro
and in rat lung in vivo
in response to CS exposure (21
). This was associated with increased NF-κB activation and acetylation of histone proteins (25
). However, the molecular mechanisms of CS-mediated lung inflammation, particularly in patients with COPD, are not completely understood. This study was focused on the role of class III deacetylases, SIRT1, which is an important protein involved in deacetylation of proteins/histones, regulation of proinflammatory cytokine release, apoptosis, stress resistance, metabolism, senescence, differentiation, and aging (19
)—all of which are linked to the pathogenesis of COPD (4
). The levels of SIRT1 were decreased in peripheral lungs, particularly in alveolar macrophages, airway epithelium, and in alveolar epithelium of smokers and patients with COPD as compared with nonsmokers. Because SIRT1 is involved in the regulation of NF-κB (19
), the decreased levels of SIRT1 may result in an NF-κB–mediated abnormal chronic inflammatory effect, which is observed in lungs of smokers and patients with COPD. Consistent with this notion, we observed the decreased levels of SIRT1 and increased activation of RelA/p65 in peripheral lungs of smokers and patients with COPD. The importance of SIRT1 further gains credence from the observation of McBurney and colleagues (37
) that genetic ablation of SIRT1 leads to increased neutrophil infiltration in mouse lung, suggesting that knockdown of SIRT1 leads to exaggerated lung inflammation. Therefore, it is possible that CS-mediated reduction in SIRT1 may in part be responsible for increased neutrophil influx, NF-κB activation, and inflammatory response seen in lungs of smokers and patients with COPD.
We further determined the molecular mechanism of SIRT1 reduction and its role in proinflammatory cytokine release in response to CS exposure in human macrophage-like cells (MonoMac6) in vitro
. Alveolar macrophages are considered to be important cells in perpetuating the inflammatory response of CS (6
). CSE treatment significantly increased the release of proinflammatory cytokine IL-8 concomitant with decreased levels of SIRT1 protein and mRNA expression in MonoMac6 cells. Recently, Yang and coworkers (21
) reported that CS-mediated decrease in SIRT1 levels was correlated with decreased SIRT1 activity in macrophages and rat lungs. Because this decrease was correlated with increased release of IL-8, we speculated that CSE-mediated decrease in the levels of SIRT1 plays an important role in release of proinflammatory cytokines. To support this observation and to determine the specific effect of SIRT1 in CS-induced proinflammatory cytokine release, further experiments were performed on MonoMac6 cells by knocking down SIRT1 or overexpressing SIRT1 as well as using a SIRT1 defective mutant lacking deacetylase activity. Knockdown of endogenous SIRT1 and mutation of SIRT1 deacetylase domain augmented the CS-stimulated proinflammatory cytokine (IL-8) release, whereas SIRT1 overexpression resulted in decreased IL-8 release in response to CSE exposure. We have previously shown that pharmacologic activation of SIRT1 reduced the CSE-mediated IL-8 release in MonoMac6 cells (21
) and the present findings further support these observations and emphasize the importance of SIRT1 in regulation of proinflammatory mediators, such as IL-8 and other NF-κB–dependent genes (matrix metalloproteinases, growth factors, and mucin genes). The mechanism whereby CS alters the levels of SIRT1 is not known, but it is possible that SIRT1 is regulated by post-translational modifications and/or by kinase signaling mechanisms. The other possible mechanism would be nucleocytoplasmic shuttling of SIRT1 by kinase signaling mechanism leading to proteasomal degradation of SIRT1 in the cytoplasm.
It is well known that CS-induced oxidative stress is responsible for proinflammatory cytokine release in the lung (39
). Previously, we have shown that levels of lipid peroxidation products, such as 4-HNE, were increased in lungs of patients with COPD (40
). Post-translational modifications of various proteins by oxidative/nitrosative stress have been shown to have influence on various cellular functions (25
). To determine the CS-mediated reduction in SIRT1 levels and its post-translational modifications, SIRT1 modification was evaluated by measuring the SIRT1 adducts with 4-HNE (reactive aldehydes which form protein carbonyls), a highly reactive diffusible product of lipid peroxidation and a key mediator of oxidant-induced cell signaling and apoptosis (43
). SIRT1 adducts with 4-HNE and 3-nitrotyrosine in lungs were increased in smokers and patients with COPD compared with nonsmokers. CSE induced the formation of SIRT1–4-HNE adducts in MonoMac6 cells. Cysteine, histidine, and lysine, the three nucleophilic amino acids, have been shown to be the target of modification by 4-HNE (43
). The high affinity of 4-HNE toward lysine becomes important with respect to SIRT1, because lysine residues 1020/1024 present on the active site domain of SIRT1 may be the direct target of 4-HNE. Thus, the increased SIRT1–4-HNE adduct formation seen after smoke exposure may therefore form a part of the mechanism responsible for the reduction in SIRT1 activity/level. However, it may also be possible that other residues, such as cysteine(s), present on SIRT1 are involved in formation of SIRT1–4-HNE adducts. Both oxidation and nitration can damage proteins; nitration of protein tyrosine residues to form 3-nitrotyrosine is considered a hallmark of tissue injury caused by inflammation (41
). Post-translationally modified proteins can be a direct target of proteolytic degradation and removal (44
). The increased SIRT1 protein tyrosine nitration seen after CSE exposure in MonoMac6 cells may trigger increased proteolytic degradation of this protein, resulting in decreased SIRT1 levels. Thus, the decreased SIRT1 levels in smokers and patients with COPD may be explained on the basis of the CS-mediated oxidative/nitrosative (which occurs in patients with COPD) alterations on the SIRT1 proteins. In view of the fact that SIRT1 is an antiaging and antiinflammatory molecule (9
), the CS-mediated SIRT1 modification/reduction may have a role in lung inflammation and aging seen in patients with COPD (3
). However, it remains to be determined whether SIRT1 reduction is directly associated with the decline in lung function in smokers or disease progression/severity of COPD. Because some of the patients with COPD were ex-smokers and some of them were receiving inhaled steroids, it is likely that, once initiated, the alterations of SIRT1 may not be fully reversible (irreversible epigenetic events), which in turn might be one contributor to the persistence of many inflammatory changes observed even after cessation of smoking.
Reduction/inactivation of other deacetylases, such as HDACs, has been reported to cause transactivation of NF-κB and induction of proinflammatory cytokine release (25
). Recently, Yeung and colleagues (19
) demonstrated that SIRT1 physically interacts with the RelA/p65 subunit of NF-κB and inhibits gene transcription by deacetylating RelA/p65 at lysine 310, suggesting that acetylated lysine 310 might form a platform for the binding of a bromodomain-containing protein that is required for full transcriptional activity of RelA/p65 (45
). Because acetylation at lysine 310 is required for full transactivation function of RelA/p65 (32
), the levels of acetylated lysine 310 moiety of RelA/p65 subunit of NF-κB in CSE-treated MonoMac6 cells were determined. It was found that CS-mediated SIRT1 reduction was associated with increased acetylation of lysine 310 moiety on RelA/p65. SIRT1 knockdown also increased the acetylation of RelA/p65 (K310) acetylation and this effect was further augmented by the acetylation effect of CS on RelA/p65. These data indicate that CSE caused acetylation of RelA/p65 NF-κB by reduction of SIRT1 deacetylase level and/or post-translational modifications of its lysine residues. Therefore, increased post-translational modifications of SIRT1 lead to disruption of RelA/p65-SIRT1 complex, which would then culminate into NF-κB acetylation and persistent activation of NF-κB in response to CSE exposure in macrophages. Apart from NF-κB regulation, SIRT1 also regulates the stress/protective pathway via deacetylation of the forkhead box class (FOXO3) transcription factor. SIRT1 reduction leads to acetylation of FOXO3, which would then result in loss of its transcription activity for transcription of GADD45 (DNA repair) and MnSOD genes (9
). Therefore, loss of SIRT1 by CS will lead to acetylation of FOXO3 and tumor suppressor p53, resulting in lung cell senescence and apoptosis.
In conclusion, we have shown for the first time that the level of nuclear SIRT1 protein was decreased in peripheral lung tissue of smokers and patients with COPD. SIRT1 proteins undergo post-translational covalent modifications in response to CS exposure, which may not be fully reversible. These changes render SIRT1 inactive, leading to acetylation/activation of RelA/p65, and thereby uncontrolled expression of proinflammatory mediators, which is seen in macrophages/lungs of smokers and patients with COPD. In view of the role of SIRT1 in regulation of proinflammatory mediators, apoptosis, senescence, cell survival, differentiation, and aging, it is tempting to propose that CS-mediated alterations in SIRT1 would have ramifications on these processes that are directly linked to the pathogenesis of COPD (4
). Further studies are required to understand the mechanism of CS-mediated down-regulation of SIRT1 and its involvement in chronic inflammatory and injurious processes in the lung using genetic gain and loss of function, and whether up-regulation or genetic modifications of SIRT1 can attenuate such processes in animal models of COPD.