Exposure to CS is the primary factor associated with the development of COPD. Although the exact mechanism leading to the development of COPD has not been fully explained, mounting evidence indicates that oxidative stress plays a significant role (5
). Since airways are continuously exposed to high levels of environmental oxidants, they must maintain the proper balance between oxidants and antioxidants to prevent cellular damage. Environmental toxicants, such as CS, that alter the cellular redox balance in the lung promote oxidative stress and over time lead to pulmonary injury and tissue damage.
CS creates an oxidant imbalance through two mechanisms. First, a burning cigarette releases millions of free radicals and increases the level of ROS and RNS in the lung. The release of these oxidants into the lungs of smokers overwhelms the normal antioxidant balance, which results in a subsequent decrease in antioxidant levels, thereby promoting oxidative stress. Xenobiotics are naturally detoxified through Phase I and Phase II enzymes, which modify xenobiotic toxicants through either oxidation or reduction of compounds (Phase I) and then promote the conjugation of phase I products with various hydrophilic moieties, including glutathione, to be safely secreted (Phase II) (43
). Numerous Phase I and Phase II detoxification enzymes as well as enzymes associated with antioxidant metabolism are induced in the lung after exposure to CS through the master transcriptional regulator, Nrf2 (18
). Indeed, chronic exposure to CS increases the expression of many Nrf2-dependent antioxidant and detoxification enzymes in airway epithelium (44
). Therefore, to combat oxidative stress generated by CS, increasing antioxidant and detoxification Nrf2-dependent pathways may have therapeutic benefits.
Nrf2-dependent pathways can be activated either pharmacologically with the use of small triterpenoid molecules such as CDDO-Im (23
) or genetically with tissue-specific Keap1 knockout mice as described in this and other studies (26
). CDDO-Im is a potent synthetic triterpenoid, which is able to increase cytoprotective gene expression in the liver, lung, and other organs (45
). This small molecule has been shown to be protective against LPS-induced inflammation and mortality by increasing antioxidant gene expression and decreasing ROS production and proinflammatory cytokine expression (46
). More recently, CDDO-Im has been shown to be protective against both pulmonary CS-induced emphysema and cardiac dysfunction by decreasing oxidative stress and cellular apoptosis in the lung in an Nrf2-dependent manner (23
). However, it is unclear whether selective targeting of Keap1
and enhancement of Nrf2 activation in the lungs will be sufficient to protect against the CS-induced oxidative stress and inflammation.
The present study is the first to report the creation of lung cell–specific Keap1 knockout mice in which Nrf2-dependent pathways are activated and more than 50 antioxidant and detoxification enzymes are increased in expression. Similar to the hepatocyte-specific Keap1 knockout mice, HO-1
, which are well-known Nrf2-dependent genes, were not significantly up-regulated in the lungs of Keap1Δ2–3/Δ2–3
mice (). The inability to induce the expression of these genes in airway epithelium may be due to the lack of other signaling events required to induce the expression of certain Nrf2-dependent genes, such as the recruitment of co-activators to the ARE (26
). Alternatively, the inability to up-regulate HO-1
may be due to the presence of a repressor protein such as Bach-1 that binds to the OH-1
promoter and inhibits Nrf2-dependent transcription (47
). A significant difference between the current study and the hepatocyte-specific Keap1 knockout mouse model is that Gclm
was significantly up-regulated in the lungs of Keap1Δ2–3/Δ2–3
mice but was not induced in the liver. This may indicate that cellular factors within different cell types influence which Nrf2-dependent genes are expressed in cells lacking a functional Keap1 protein.
Interestingly, a subset of cytoprotective and detoxification genes were significantly decreased in the lungs of Keap1Δ2–3/Δ2–3;CctCre+ mice. Many of these genes were associated with the innate or adaptive immune response such as Cybb and Gzma, respectively. Since Keap1 is deleted from airway epithelial cells only and the microarray used tissue from the whole lung, it may be possible that certain genes are down-regulated as a compensatory mechanism to maintain homeostasis in the lung. Indeed, no difference existed between the lung structure of Keap1flox/flox mice and Keap1Δ2–3/Δ2–3;CctCre+ mice. Therefore, increasing Nrf2-dependent gene expression in Clara cells may have downstream effects in other pulmonary cell types.
Corresponding to the increase in antioxidant gene expression, Clara cells isolated from Keap1Δ2–3/Δ2–3;CctCre+ mice had decreased levels of intracellular ROS after the induction of oxidative stress (e.g., hydrogen peroxide). Keap1Δ2–3/Δ2–3;CctCre+ mice also had significantly increased levels of total GSH in the lungs during normal unstressed conditions as well as immediately after CS exposure. Recruitment of proinflammatory macrophages as a result of CS was blunted in Keap1Δ2–3/Δ2–3;CctCre+ mice, indicating that the increases in antioxidant and detoxification enzymes in the lung are beneficial to suppress inflammation.
The deletion of exons 2 and 3 of the Keap1 gene and elimination of the IVR domain and Kelch domains 1 through 4 in this novel mouse model led to a decrease in Keap1
gene expression and protein levels and an increase in Nrf2-dependent gene expression. No truncated Keap1 protein was detected in the lungs of Keap1Δ2–3/Δ2–3
mice. Therefore, we hypothesize that the truncated Keap1 protein in the conditional knockout mice is rapidly degraded through the proteasomal pathway. This hypothesis is supported by studies in which mutations in KEAP1 have been identified in a significant percentage of primary human lung cancer tumors and cell lines (33
). Mutations in functionally important domains of KEAP
1 in human non–small cell lung cancer cell lines decrease the protein levels of KEAP1 compared with nonmalignant cells (33
). The loss of function in Keap1 and gain of Nrf2 function is not always beneficial and may promote tumor progression. However, the lungs of Keap1Δ2–3/Δ2–3
mice were analyzed at 8 weeks of age and have MLI and surface to volume ratios similar to the floxed control mice, indicating that the disruption of Keap1
in airway epithelial cells does not alter lung structure. In addition, Keap1Δ2–3/Δ2–3
mice greater than 6 months of age have no observable tumors or pathologies in the lung, suggesting that other mutations in addition to the deletion of Keap1 is necessary to trigger the development of lung cancer.
In summary, a novel Clara cell–specific Keap1 knockout mouse model was generated that had a significant increase in many Nrf2-dependent antioxidant and phase II detoxification enzymes in the lung. Clara cells from Keap1Δ2–3/Δ2–3;CctCre+ mice were protected against ROS in response to oxidative stress as a result of increased expression of Nrf2 pathways. In the lungs deletion of Keap1 led to a significantly increased level of total GSH that remained elevated after exposure to CS. The increased antioxidant levels correlated with an attenuation of inflammatory macrophages as well as dampened expression of MCP-1 after acute CS exposure. Increased expression of Nrf2 pathways in Clara cells may therefore protect against lung destruction due to chronic exposure to CS exposure.