FoxO3 has been shown to regulate expression of several genes involved in inflammation, oxidative stress resistance, and senescence which are intertwined in the pathogenesis of COPD. However, the role of FoxO3 in lung inflammation and pathogenesis of COPD/emphysema is not known. We found that the abundance of FoxO3 was decreased in lungs of smokers, and even more prominently reduced in patients with COPD compared to nonsmokers. FoxO3 was also reduced in mouse lungs in response to CS exposure, which is a major risk factor in development of COPD. Furthermore, the deficiency in FoxO3 increased the susceptibility to CS-induced emphysema, which was reflected by airspace enlargement, impaired lung function, and decreased exercise performance. All these findings suggest CS-induced reduction of FoxO3 is a key contributing factor in development of COPD/emphysema. The mechanism underlying CS-induced reduction of FoxO3 is unclear, but it may be associated with its post-translational modifications, such as acetylation and phosphorylation. This is corroborated by findings that increased acetylation of FoxO3 occurred in lungs of smokers and mouse exposed to CS.
Sustained inflammation is a characteristic feature in pathogenesis of COPD/emphysema, and FoxO3 is known to be involved in augmented inflammatory responses (18
). Therefore, we determined whether susceptibility of FoxO3-/-
mice to development of emphysema is associated with increased lung inflammatory response. CS exposure increased inflammatory cell infiltration into the lung accompanied with proinflammatory cytokines release, including MCP-1, KC and IP-10 in WT mice, which was more pronounced in lungs of FoxO3-/-
mice. This finding is consistent with a previous study showing that an enhanced inflammatory response occurred in intestinal tissue of FoxO3-/-
mice challenged with dextran sulfate sodium as compared to WT mice (55
). Hence, increased inflammatory response may contribute to susceptibility in development of emphysema in FoxO3-/-
FoxO3 was predominantly localized in both lung airway/alveolar epithelium and macrophages in nonsmokers, and significantly decreased in all locations in lungs of smokers and patients with COPD. These findings raise a question as to which specific cell-type is responsible for CS-induced pathological changes (e.g.
inflammation) in lungs of FoxO3-/-
mice. We, therefore generated the BMT chimeric mice in order to distinguish the role of FoxO3 in lung epithelial cells (radioresistant non-hematopoietic-derived structural cells) and inflammatory cells (radiosensitive hematopoietic-derived cells) in CS-induced lung inflammation. We found that CS-induced release of proinflammatory cytokines was increased in FoxO3-deficient mice in hematopoietic or non-hematopoietic cells as compared to the mice expressing FoxO3 in these cells. Deficiency in FoxO3 in both the cells further increased the release of proinflammatory mediators in response to CS exposure, suggesting that both structural cells (mainly epithelial cells) and hematopoietic cells (mainly macrophages and neutrophils) are responsible for the altered responses to CS exposure seen in FoxO3-/-
mice. Although it has been reported that simultaneous conditional deletion of FoxO1, FoxO3 and FoxO4 results in long-term defect of hemotopoietic stem cell (56
), Miyamoto et al.
has demonstrated that young adult FoxO3-/-
mice (8- to 12 week-old) show normal proliferation and differentiation of hematopoietic progenitors, which is supported by normal morphology and cell numbers in lymphoid and myeloid cells in peripheral blood and bone marrow (57
). Consistent with the latter observation, we did not find any differences in differential cell counts from myeloid cell-derived neutrophils and macrophages in air-exposed FoxO3-/-
and BMT chimeric mice. A recent study has also shown that the loss of FoxO3 alone does not alter intrinsic phenotype and activation of T cell (18
Although we did not investigate the lymphoid cell population in BMT chimeric mice, CD8+ T cell product, IP-10 levels were similar in between air-exposed chimeric mice (unpublished observations), possibly suggesting that lymphoid-lineage cells of hematopoiesis is not impaired in FoxO3-/- mice. As evidenced by a significant increase of IP-10 levels (a specific chemoattractant for activated T cells), shown in chronic CS exposure, BMT chimeric mice exposed to chronic CS may broaden the understanding of the role of FoxO3 in regulation of immune response by CS exposure and in pathogenesis of COPD.
It has been shown that CS induces lung inflammation which is accompanied by NF-κB activation in experimental mouse model, and IκB kinase which is implicated in activation of NF-κB regulates FoxO3 activity (25
). Therefore, we postulated that increased susceptibility to CS-induced inflammation in FoxO3-/-
mice was due to exaggerated increase in NF-κB activity. Interestingly, we found that CS exposure led to increased NF-κB RelA/p65 DNA binding activity in WT mice, which was augmented in FoxO3-/-
mice. These data suggest that NF-κB activity is inhibited by FoxO3, which is consistent with a previous report that FoxO3 can function as NF-κB antagonist and inhibit its activation (36
). In addition, we demonstrated the FoxO3-RelA/p65 interaction in response to CS in lung in vivo
and in epithelial cells in vitro
. It may be possible that FoxO3-RelA/p65 interaction specifically affects RelA/p65 activity by blocking its DNA binding in response to CS. Recent data showed that N-terminal region of FoxO4 interacts with Rel-homology domain of NF-κB, which was confirmed by a series of deletion mutants of FoxO4 and NF-κB (59
). Since forkhead DNA-binding domain was included in the N-terminal region of FoxO4 as well as FoxO3, FoxO3 may bind directly with NF-κB through its forkhead domain. However, further studies are required to identify the interactive domain between FoxO3 and NF-κB, and their involvement in control of inflammatory processes.
Since it has been known that RelA/p65 translocated into the nucleus in response to oxidative stress and CS, we speculated that CS also induced nuclear translocation of FoxO3 to interact with RelA/p65. Brunet et al.
reported that FoxO3 was localized in cytoplasm when growth factors were present, and translocated into the nucleus in response to oxidative stress (30
). Consistent with this observation, we found that CS/oxidative stress promoted FoxO3 translocation into the nucleus in vivo
in the lung and in vitro
in bronchial epithelial cells. Although the phosphorylation of FoxO3 causes proteolysis of FoxO3 via ubiqutin-proteasome pathway in the cytoplasm (25
), a recent study reported that oxidative stress-induced de-phosphorylation of FoxO3 promoted nuclear translocation of FoxO3 without affecting the total FoxO3 levels (60
). Similarly, we also found that CSE induced de-phosphorylation of FoxO3 in a time-dependent fashion in bronchial epithelial cells (data not shown), which was accompanied with nuclear translocation of FoxO3. The mechanism underlying CS-induced de-phosphorylation and translocation of FoxO3 into the nucleus is not known. However, it may be possible that CS-induced de-phosphorylation of FoxO3 is closely related with its translocation into the nucleus. Oxidative stress due to imbalance between oxidants and antioxidants is involved in pathogenesis of chronic pulmonary diseases (61
). It has been reported that FoxO3 plays a role in regulation of cellular ROS levels through modulation of transcription of antioxidant genes (22
). In this study, we showed that the mRNA and protein expression of MnSOD and catalase, which are well-known specific target genes of FoxO3, were increased in CS-exposed WT mice, whereas the levels were significantly reduced in CS-exposed FoxO3-/-
mice at 3 days. Despite reduction of FoxO3 in lungs of CS-exposed WT mice, the expression of its target MnSOD and catalase was increased in CS-exposed WT mice. This discrepancy may be due to increased acetylation of FoxO3 against acute lung inflammation. We showed that acetylation of FoxO3 was increased in lungs of smokers, as well as in lungs of mice exposed to CS suggesting alteration in transactivation of FoxO3 for target genes, such as MnSOD and catalase. CS-induced oxidant stress may potentially modify FoxO3 via CBP-mediated acetylation which is shown to be the case for FoxO4 (64
) and SIRT1-mediated deacetylation (30
). We have recently shown that the levels of SIRT1 are reduced in lungs of smokers and patients with COPD as well as in mouse lung (33
), which can result in acetylation of FoxO3. Transcriptional activity of Foxo3 is modified mainly by post-translational modifications and association with many different cofactors (65
). Although many studies have described that post-translational modifications, such as phosphorylation and acetylation, of FoxO3 lead to the repression of its transcriptional activity, some suggested that acetylation of FoxO3 increases the target gene transcription (30
). Therefore, it is possible that CS-induced acetylation of FoxO3 enhances the transcription of antioxidants genes to counteract oxidative stress. In 4 months of CS exposure, however, the expression of antioxidant genes was reduced both in CS-exposed WT and FoxO3-/-
mice due to the loss of FoxO3 levels per se
. Altogether, our data suggest that acetylation of FoxO3 may enhance transcription of antioxidant genes but further study is required to assess the role of acetylation and deacetylation of FoxO3 in regulation of MnSOD and catalase under the condition of oxidative stress.
In conclusion, we show a novel role of FoxO3 in regulation of lung inflammation and in pathogenesis of COPD/emphysema. The abundance of FoxO3 was decreased in lungs of patients with COPD. Furthermore, genetic ablation of FoxO3 in mice led to increased susceptibility to airspace enlargement/emphysema in response to CS, in association with exaggerated lung inflammatory response and decreased antioxidant genes due to FoxO3 deficiency. We further provide a novel mechanistic link between FoxO3 and NF-κB both in vivo and in vitro, and suggest that FoxO3 acts as a fine tuner that modulates CS-induced lung inflammatory response and COPD/emphysema. In addition, it may be possible that cellular senescence/accelerated lung aging which are important events in pathogenesis of COPD are regulated by FoxO3 via SIRT1 deacetylase. Hence, further studies are required to expand our understanding for the role of FoxO3 in pathogenesis of COPD/emphysema particularly with respect to regulation of antioxidant defense mechanisms and cellular senescence in the lung.