Distal airways in mice exposed to cigarette smoke: Nrf2-regulated genes are increased in Clara cells

Abstract

Cigarette smoke (CS) is the main risk factor for chronic obstructive pulmonary disease (COPD). Terminal bronchioles are critical zones in the pathophysiology of COPD, but little is known about the cellular and molecular changes that occur in cells lining terminal bronchioles in response to CS. We subjected C57BL/6 mice to CS (6 d/wk, up to 6 mo), looked for morphologic changes lining the terminal bronchioles, and used laser capture microdissection to selectively isolate cells in terminal bronchioles to study gene expression. Morphologic and immunohistochemical analyses showed that Clara cell predominance remained despite 6 months of CS exposure. Since Clara cells have a role in protection against oxidative stress, we focused on the expression of antioxidant/detoxification genes using microarray analysis. Of the 35 antioxidant/detoxification genes with at least 2.5-fold increased expression in response to 6 months of CS exposure, 21 were NF-E2-related factor 2 (Nrf2)-regulated genes. Among these were cytochrome P450 1b1, glutathione reductase, thioredoxin reductase, and members of the glutathione S-transferase family, as well as Nrf2 itself. In vitro studies using immortalized murine Clara cells (C22) showed that CS induced the stabilization and nuclear translocation of Nrf2, which correlated with the induction of antioxidant and detoxification genes. Furthermore, decreasing Nrf2 expression by siRNA resulted in a corresponding decrease in CS-induced expression of several antioxidant and detoxification genes by C22 cells. These data suggest that the protective response by Clara cells to CS exposure is predominantly regulated by the transcription factor Nrf2.

Figure 1.

Chronic cigarette smoke (CS) exposure induces flattening of Clara cells in terminal bronchioles. Scanning electron microscopy of lungs harvested from C57BL/6 mice exposed to room air (A and C) or CS (B and D) for 6 months. Higher magnifications are shown in C and D. Images are representative of five terminal airways per mouse from five mice per condition.

Figure 2.

CS does not alter cell phenotype in the terminal bronchiolar region. (A) Morphometric and (B) immunohistochemical analyses of the terminal bronchioles of mice exposed to room air (nonsmoking) or CS for 6 months. Ten terminal airways per mouse from five mice per condition were evaluated. Results are expressed as an average number of each cell type per terminal airway ± SEM.

Figure 3.

Chronic CS exposure increases Nrf2 expression in terminal bronchiolar epithelium. (A) RNAs isolated from laser capture microdissection–retrieved cells from terminal bronchiolar region of mice exposed to CS for various times were subjected to real-time RT-PCR using oligonucleotides specific for murine Nrf2. Data are representative of three separate experiments using two independently isolated RNA samples per time point. Expression of Nrf2 was normalized to that of the β2-MG housekeeping gene ± SEM. *P < 0.005 relative to nonsmoked control. Sections of lungs from mice exposed to (B) room air, (C) a single CS exposure, or (D) CS repeatedly for 6 months were immunostained using an anti-Nrf2 antibody. (E) Secondary antibody alone served as negative control. Images are representative of four mice per condition.

Figure 4.

CS extract directly induces oxidative stress in C22 cells. (A) C22 cells were exposed to serum-free media alone or containing various amounts of CS extract (CSE) for 24 hours, and the total antioxidant potential of the cells was determined. Data are representative of three independent experiments performed in triplicate ± SEM. *P < 0.005; **P < 0.05 relative to 0% CS extract. C22 cells were exposed to serum-free media alone (B and D) or media containing 10% CS extract (C and E) for 24 hours and stained for oxidative DNA damage using an anti–8-OHdG antibody (B and C). Nuclei were detected with DAPI (D and E).

Figure 5.

CS extract induces C22 cell expression of several antioxidant and detoxification genes. RNAs isolated from C22 cells exposed to serum-free media alone (open bars) or containing 10% CS extract (solid bars) for 3 hours were subjected to real-time RT-PCR using gene-specific oligonucleotides for several (A) antioxidant and (B) detoxification genes. After normalization to β2-MG, results are expressed as fold change above untreated conditions. Data are representative of three independent experiments ± SEM.

Figure 6.

Exposure of C22 cells to CS extract results in Nrf2 translocation to the nucleus and stabilization, but does not increase Nrf2 expression. (A) RNA isolated from C22 cells exposed to serum-free media alone or containing 10% CS extract for up to 24 hours were subjected to real-time RT-PCR using Nrf2-specific oligonucleotides. After normalization to β2-MG, results are expressed as fold change above untreated conditions. Data are representative of three independent experiments ± SEM. (B) Western blot analysis of whole cell protein extracts collected from C22 cells exposed to CS for up to 24 hours using an Nrf2-specific antibody. The same membrane was also probed with a GAPDH-specific antibody to control for loading. Data are representative of at least three independent experiments. (C) C22 cells were exposed to serum-free media containing 10% CS extract for up to 1 hour. Cells were fixed and stained using a Nrf2-specific antibody. (D) C22 cells were exposed to serum-free media containing 10% CS extract for up to 1 hour and the amount of Nrf2 located in the cytoplasmic (C) and nuclear (N) fractions was determined by Western blot analysis using an Nrf2-specific antibody. Data are representative of at least three independent experiments.

Figure 7.

CS-induced antioxidant and detoxification gene expression is primarily regulated by the Nrf2 transcription factor. (A) C22 cells were transfected with siRNA duplexes targeting the expression of the Nrf2 or NF-κB transcription factors, or a scrambled siRNA duplex. Two days after transfection, RNAs were isolated and subjected to real-time RT-PCR for expression of Nrf2 or NF-κB, respectively. Data are representative of at least three independent experiments ± SEM. *P < 0.005 relative to nontransfected control. (B) In separate experiments, 2 days after transfection with scrambled (Scr, open squares), Nrf2 (solid squares), or NF-κB (shaded squares) siRNA duplexes, C22 cells were exposed to serum-free media containing 0–10% CS extract for 24 hours and the relative amount of lactate dehydrogenase (LDH) in the media was determined. Results are expressed as fold change relative to C22 cells that were transfected with the Scr siRNA, exposed to serum-free media alone. (C and D) Two days after transfection with Scr (open bars), Nrf2 (solid bars), or NF-κB (shaded bars) siRNA duplexes, C22 cells were exposed to serum-free media containing 10% CS extract for 3 hours. RNAs were isolated and subjected to real-time RT-PCR using gene-specific oligonucleotides for several (C) antioxidant and (D) detoxification genes. After normalization to β2-MG, results are expressed as fold change relative to C22 cells transfected with the Scr siRNA. Data are representative of at least three independent experiments ± SEM. *P < 0.005; **P < 0.05 relative to scramble siRNA control.