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Published ahead of print on November 1, 2006, doi:10.1165/rcmb.2006-0248OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 36, pp. 418-426, 2007
© 2007 American Thoracic Society
DOI: 10.1165/rcmb.2006-0248OC

Enhanced Expression of MafB Inhibits Macrophage Apoptosis Induced by Cigarette Smoke Exposure

Jun-ichi Machiya, Yoko Shibata, Keiko Yamauchi, Noriyuki Hirama, Toshihiro Wada, Sumito Inoue, Shuichi Abe, Noriaki Takabatake, Makoto Sata and Isao Kubota

Department of Cardiology, Pulmonology, and Nephrology, Yamagata University School of Medicine, Yamagata, Japan

Correspondence and requests for reprints should be addressed to Yoko Shibata, M.D., Ph.D., Department of Cardiology, Pulmonology, and Nephrology, Yamagata University School of Medicine, 2-2-2 Iida-Nishi, Yamagata 990–9585, Japan. E-mail: shibata{at}med.id.yamagata-u.ac.jp


   Abstract
Top
 Abstract
CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
In the lungs of smokers, oxidative stress rises due to increase of free radicals and oxidants, including lipid peroxide (LPO). The functions of alveolar macrophages (AMs) are altered in such an environment, and their survival is prolonged against toxicities of cigarette smoke (CS) by an unknown mechanism. Whereas functions of AMs are potentially regulated by various transcriptional factors, their expressions and roles in smoking individuals have not been elucidated. Therefore, we investigated their expressions using murine model of CS exposure. Eight-week-old male B6C3F1 mice were whole-bodily exposed to CS (2 cigarettes/mouse/day, 5 d/wk) for 6 mo. Development of pulmonary emphysema in 6-mo CS-exposed mice was confirmed by a morphometric analysis. Among the transcriptional factors investigated, only MafB was upregulated in AMs from CS-exposed mice. DNA binding capacity of MafB for Maf recognition element was also increased in AMs from those mice. LPO was increased significantly in the lungs of CS-exposed mice. Because the end product of LPO, 4-hydroxy-2-nonenal, enhanced MafB expression and its transcriptional activity in a cultured macrophage cell line, LPO-related oxidative stress was suggested to be involved in the mechanism of MafB expression in CS-exposed lung. Furthermore, we established a macrophage cell line that can overexpress MafB and thereby clarify the role of MafB. Forced expression of MafB heightened cell viability and attenuated the occurrence of apoptosis in cells treated with CS-extract. These results suggest that enhanced MafB expression by oxidative stress inhibits AM cell death and prolongs their survival in the CS-exposed lung.

Key Words: alveolar macrophages • apoptosis • transcription factors • MafB • cigarette smoking



   CLINICAL RELEVANCE
Top
 Abstract
 CLINICAL RELEVANCE
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
In this research, we present the expression and potential role of macrophage-specific transcription factor MafB in alveolar macrophages of smoke-exposed lungs. We demonstrate a new mechanism governing macrophage survival in the smoking lung via MafB.

 
Cigarette smoke (CS) is an important risk factor for many lung diseases, including chronic obstructive pulmonary disease (COPD). CS consists of more than 4,700 chemical compounds, of which both tar and gas phases contain high concentrations of free radicals and other oxidants (1). CS is also a source of highly reactive aldehydes, which are generated endogenously during lipid peroxidation (2). In the alveolar space of smokers' lungs, oxidative stress rises due to the increased release of reactive oxygen species such as superoxide anion and hydrogen peroxide from alveolar macrophages (AMs) (3, 4). Consequently, CS exerts many toxic effects on AMs and other lung cells.

Previously, Aoshiba and coworkers demonstrated that CS induced apoptosis in AMs through mitochondrial dysfunction, not through caspase activation, in vitro (5). Nevertheless, CS is widely believed to increase the number of AMs and prolong the survival of AMs in vivo (6–8). The mechanisms through which CS heightens the viability of AMs in smokers' lungs despite its proapoptotic bioactivity are poorly understood.

The functions of AMs in the smoking individuals are thought to be altered by oxidants and chemicals in CS (9). The functions of macrophages are regulated by various transcriptional factors (TFs) (10). Gene-targeted experiments have demonstrated the important roles of PU.1, CCAAT enhancer-binding protein (C/EBP)-{alpha}, and interferon regulatory factors (IRFs) for macrophage functions (10, 11). In cases in which expression of TF is altered, the functions of AMs are changed drastically, as we reported previously (11). Thus, the prolonged survival of AMs in smokers may be attributable to alteration of TF's activity in AMs.

The TF MafB is an inducer of monocytic differentiation (12). The Maf family proteins possess a basic leucine zipper structure at the carboxyl terminus, which mediates dimer formation and DNA binding to the Maf recognition element (MARE) (13). The Maf family proteins are classified into two subfamilies: the large Maf and the small Maf group. Large Maf proteins such as MafB possess an N-terminal acidic domain, which serves as a transactivation domain, whereas small Maf proteins defect it. MafB has been identified as a protooncogene responsible for multiple myeloma (14, 15), where it might keep cells viable by inhibiting apoptosis (16). Although MafB regulates the functions of AMs (12), the expression of MafB in AMs of CS-exposed lungs has not been investigated.

This study evaluated the expressions of MafB in an experimental mouse model of CS-exposure. Our data showed marked up-regulation of MafB by CS exposure. Thus, we hypothesized that MafB could maintain the macrophage viability and prevent apoptosis induced by CS. To address the potential role of MafB for macrophage survival, we established a MafB-overexpressing macrophage cell line and analyzed the functions of MafB in vitro.


   MATERIALS AND METHODS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
CS Exposure
Eight-week-old male B6C3F1 mice, of an extremely CS-sensitive strain for development of pulmonary emphysema (17), were whole-bodily exposed to smoke from commercial nonfiltered cigarettes (2 cigarettes/mouse/day, 5 d/wk, Peace; Japan Tobacco Inc., Tokyo, Japan) using a whole-body smoking exposure apparatus (INH03-CIGR01A; MIPS, Osaka, Japan) for 1, 3, 7, 14, or 180 d (17). Control mice were not exposed to CS. The animals were handled according to the animal welfare regulations of Yamagata University. The Animal Subjects Committee of Yamagata University approved the study protocol. Furthermore, this investigation conformed to the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health.

Lung Fixation and Morphometry
At 24 h after the final exposure of CS, seven mice of each group were killed using pentobarbital. The lungs were excised and fixed intratracheally with buffered formalin (10%) at a constant pressure of 25 cm H2O to prepare the paraffin-embedded lung blocks; subsequently, the sections were stained using hematoxylin-eosin. Digitized images of the lung fields were taken using a digital camera (Camedia C-2000 Zoom; Olympus Optical Co. Ltd., Tokyo, Japan) that was fitted on a microscope (BX50–33-FLA2; Olympus Optical Co.). The images were analyzed using computer software (Photoshop CS; Adobe Systems Inc., Tokyo, Japan) (18). The mean linear intercept, as a measure of interalveolar septal wall distance, was measured using light microscopy at a magnification of x100. The mean linear intercept was obtained by dividing the length of a line drawn across the lung section by the total number of intercepts encountered in 36 lines per mouse lung, as described previously (19).

Bronchoalveolar Lavage Procedure
A 20-gauge catheter was inserted into the trachea and bronchoalveolar lavage (BAL) was performed by instilling 10 times with 1-ml of PBS. Total cell numbers were counted using a hemacytometer. Cell differentials were counted on stained (Diff-Quick solutions; International Reagents Corp., Kobe, Japan) cytospin preparations (18).

RT-PCR
Gene expressions in BAL cells were examined using RT-PCR. Sequences of specific primers are listed in Table 1. Preliminary, PCR cycle numbers were determined within the linear amplification range. PCR products were electrophoresed in 1% agarose gels containing ethidium bromide; they were visualized digitally with an ultraviolet illuminator (AB1500 Printgraph and AE 6905H Image Saver HR; ATTO Bioscience, Tokyo, Japan). The band intensities were semiquantified using computer software (Lane Analyzer 3.0; ATTO Bioscience).


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  		TABLE 1. SEQUENCE OF PCR PRIMERS USING IN THIS STUDY

 
Immunostaining of MafB
Immunostaining was performed as described previously (18). Cytospin preparations were incubated with goat anti-MafB antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA) at 1:100 dilution and biotin-conjugated horse anti-goat IgG (Vector Laboratories, Inc., Burlingame, CA) at 1:200 dilution.

Nuclear Extract Preparation
The BAL cells were pooled from 10 CS-exposed mice or 10 control mice. Nuclear extracts were then obtained using the method described previously (20).

Electrophoretic Mobility Shift Assay
Activity of MafB was assessed by electrophoretic mobility shift assay (EMSA) with consensus oligonucleotides of MARE (GGC CTG TTG CTG ACT CAG CAG GG). We used an EMSA kit (LightShift Chemiluminescent; Pierce Biotechnology, Inc., Rockford, IL), in which biotin end-labeled DNA probes were used instead of radioactive probes (21). Nuclear protein (4 µg) was incubated with labeled probes for 20 min at room temperature. The mixture was electrophoresed on a polyacrylamide gel and transferred to a nylon membrane. The biotin end-labeled DNA was detected using the manufacturer's protocol. Band specificity was determined through competition experiments with a molar excess of unlabeled consensus oligonucleotides of MARE that were added to nuclear extracts before the addition of labeled probes. Supershift assays for MafB proteins were also performed using anti-MafB antibody. Nuclear extracts were incubated with anti-MafB antibody for 1 h at 4°C before the labeled probes were added.

Measurement of Lipid Peroxide–Related Oxidative Stress in CS-Exposed Lung
Concentrations of lipid peroxide (LPO) in the lung homogenate were measured using a LPO-586 assay kit (Oxis International, Inc., Portland, OR) according to the manufacturer's protocols.

Reporter Plasmid Constructs and Transfection into RAW264.7 Cells
A DNA fragment containing trimerized MAREs (AAT TGC TGA CTC AGC AGG GTG CTG ACT CAG CAC CCT GCT GAC TCA GCA GTC GAC AGC GGA GAC CTA GAG GGT ATA TA) was synthesized to prepare the MARE beta-lactamase reporter constructs (Invitrogen Corp., Carlsbad, CA). This DNA fragment was subcloned into pGeneBLAzer-TOPO (Invitrogen Corp.). This vector, which was designated as pMARE-Bla, was transfected into RAW264.7 cells using GenePORTER2 (Gene Therapy Systems, Inc., San Diego, CA) by the procedure recommended by the manufacturer. Stable cells were selected by resistance to 800 µg/ml geneticin, and were named as RAW264.7-pMARE-Bla cells.

Murine MafB cDNA was subcloned into a pcDNA6.2/V5/GW/D-TOPO (Invitrogen Corp.) according to manufacturer's protocol. This vector was designated as pcMafB. The sequence was confirmed using autosequence analysis. As control, an empty vector was named as pcCTRL. Using GenePORTER2, pcMafB or pcCTRL was transfected into RAW264.7-pMARE-Bla cells. Stable cells were selected by resistance to 7.5 µg/ml blasticidin, and were named respectively as RAW264.7-pMARE-Bla-pcMafB cells or RAW264.7-pMARE-Bla-pcCTRL cells.

Analysis of beta-Lactamase Reporter Assay
beta-lactamase activity was detected using an in vivo and in vitro detection kit (GeneBLAzer; Invitrogen Corp.) (22). In a population of cells loaded onto a CCF2 substrate, those that fluoresced blue contained beta-lactamase reporter activity, whereas those that fluoresced green did not (23).

The RAW264.7-pMARE-Bla cells were seeded onto six-well dishes at 1 x 106 cells/well 24 h before stimulation. Media were changed to a complete medium or medium containing 20 µM 4-hydroxy-2-nonenal (4-HNE). Cells were harvested 18 h later to obtain cell lysates for evaluation of beta-lactamase activity using a spectrofluorometer (FP-6300; Jasco Inc., Tokyo, Japan).

The RAW264.7-pMARE-Bla-pcMafB cells and RAW264.7-pMARE-Bla-pcCTRL cells were seeded onto 6-well dishes at 1 x 106 cells/well. Cells were harvested 24 h later to obtain cell lysates for beta-lactamase assay. Data were calculated as the ratio of the emissions 460/530 nm after subtraction of the background values.

Establishment of MafB-Overexpressing Macrophage Cells
Constitutive expression of murine MafB was achieved by transduction of RAW264.7 cells with a plasmid vector expressing MafB. As a transduction control, an empty plasmid, pcCTRL, was transduced into RAW264.7 cells. Antibiotic selection was started 48 h after transduction with blasticidin (7.5 µg/ml). After 10–20 d in selection medium, cells were applied to a limiting dilution method to obtain MafB-overexpressing clones. Expression of MafB in each clone was examined using RT-PCR and immunoblotting. From all 32 clones, 9 clones which expressed MafB four times as much as control cells were selected as the MafB-overexpressing cells. These cells were designated as RAW264.7-pcMafB, whereas control cells were named as RAW264.7-pcCTRL.

Immunoblotting of MafB
We applied 50 µg protein of lysates to the immunoblotting using rabbit anti-MafB (1:100 dilution, BL658; Bethyl Laboratories, Inc., Montgomery, TX) and horseradish peroxidase–conjugated anti-rabbit IgG immunoglobulin (1:6,000 dilution; Santa Cruz Biotechnology) (24).

Preparation of CS Extract
The CS extract (CSE) was produced as reported by Li and colleagues (25). Briefly, smoke from one piece of cigarette (Peace; Japan Tobacco Inc.) was bubbled through 25 ml of Hanks' Balanced Salt Solution. The resultant product was defined as 100% CSE.

Assessment of Cell Viability after CSE Exposure
The RAW264.7-pcMafB cells and RAW264.7-pcCTRL cells were seeded onto 96-well dishes at 1 x 105 cells/well and allowed to attach overnight. After the cells were treated with 20% CSE for 12 h, their viability was evaluated using an assay for a tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)- 2H-tetrazolium, inner salt; MTS] (CellTiter96 AQueous One Solution Cell Proliferation Assay; Promega Corp., Madison, WI), according to manufacturer's protocol (26). Data were adjusted using the mean absorbance value of untreated cells in each cell line.

Assessment of Apoptosis by CSE Exposure
Apoptosis by CSE exposure was evaluated using APOPercentage Apoptosis Assay Kit, which allows for the analysis of cells entering the early apoptosis (Biocolor Ltd, Northern Ireland, UK) (27) and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) method (5). For APOPercentage Apoptosis Assay, RAW264.7-pcMafB cells and RAW264.7-pcCTRL cells were seeded onto 96-well dishes at 1 x 105 cells/well. After cells were treated with 20% CSE for 6 h, the apoptosing cells were stained with the dye according to the manufacturer's protocol. The absorbance at 550 nm in the lysate was measured using a microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA) (28). For the TUNEL method, cells were seeded onto a Lab-Tek Chamber Slide (Nunc, Rochester, NY) at 1 x 105 cells/well. After cells were treated with 20% CSE for 6 h, TUNEL staining was performed using a TUNEL system (DeadEnd Colorimetric TUNEL System; Promega Corp.) according to the manufacturer's protocol.

Caspase-3 Activity
The RAW264.7-pcMafB cells and RAW264.7-pcCTRL cells were seeded onto a 6-well plate. Cells were lysed with buffered surfactant using a caspase-3 colorimetric assay kit (BioVision Inc., Mountain View, CA) 16 h after cells were treated with 0, 10, or 20% of CSE. We incubated 100 µg of the protein of lysates with p-nitroanilide-labeled caspase-3 substrate for 90 min. Caspase-3 activity was determined by measuring the absorbance at 405 nm.

Cytochrome C Releasing Assay
The RAW264.7-pcMafB cells and RAW264.7-pcCTRL cells were seeded onto 10-cm culture dishes. Proteins of cytoplasmic and mitochondrial fractions were extracted using a Cytochrome c Releasing Apoptosis Assay Kit (BioVision Inc.) 16 h after cells were treated with 0% or 20% of CSE, according to the manufacturer's protocols. The levels of cytochrome c in both fractions were evaluated by immunoblotting using cytochrome c antibody (BioVision Inc.).

Cellular Proliferation Assay
Subsequently, 1,000 RAW264.7-pcMafB cells and RAW264.7-pcCTRL cells were seeded in each well of two 96-well plates and allowed to attach overnight. Their growth rate was determined by colorimetric quantification using a Celltiter 96 Aqueous One Solution Cell Proliferation Assay Kit (Promega Corp.). Briefly, cells were incubated for 0, 24, or 48 h from the beginning of assay, after which 10 µl of MTS tetrazonium solution was added to each well; then the absorbance at 490 nm was measured. Viable cell numbers were calculated as the ratio of the absorbance value at each time point against the mean absorbance value of each cell line at 0 h.

Statistical Analyses
A Mann-Whitney U test for nonparametric data was used to analyze the difference between two groups. In figures, results are expressed as respective means ± SEM. The median values were also demonstrated in the text. Significance was inferred for results for which P < 0.05. Statistical analyses were done using computer software (Statview 5.0; SAS Institute, Inc., Cary, NC).


   RESULTS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
Expression of TFs in BAL Cells from 6-mo CS-Exposed Mice
As numerous investigators have demonstrated (29–31), we observed airspace enlargement, a typical finding of COPD, in 6-mo CS-exposed mice (Figure 1A). The values of the mean linear intercepts were increased significantly in 6-mo CS-exposed mice (Figure 1A, right; median value: control, 46.3 µm; smoking, 57.5 µm). In contrast, no development of airspace enlargement was observed in the 3-mo CS-exposed mice (data not shown). Furthermore, RT-PCR analyses for BAL cells revealed that MafB mRNA expressions were elevated in 6-mo CS-exposed mice compared with those in control mice (Figure 1B, P < 0.01; median value: control, 1.07; smoking, 6.51). In contrast, PU.1, IRF-1, IRF-2, IRF-7, and C/EBP{alpha} expressions remained unchanged (PU.1: 0.98- ± 0.12-fold increase versus control, P = 0.92; IRF-1: 0.87- ± 0.07-fold increase versus control, P = 0.040; IRF-2: 0.98- ± 0.16-fold increase versus control, P = 0.94; IRF-7: 1.73- ± 0.49-fold increase versus control, P = 0.18; C/EBP{alpha}: 0.84- ± 0.04-fold increase versus control, P = 0.13, n = 6 in each group). Elevations of MafB protein in BAL cells were confirmed by immunostaining with MafB antibody. Strong staining of MafB was observed in AMs (Figure 1C, arrowheads) from CS-exposed mice, but not in their neutrophils (Figure 1C, arrows). The yields of protein from BAL cells were so low that we could not detect MafB protein in Western blotting (data not shown).


Figure 1
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  		Figure 1. Development of pulmonary emphysema and the expression of MafB in BAL cells by chronic CS exposure. (A) B6C3F1 mice were exposed to CS for 6 mo. Microscopic observation revealed enlarged alveoli and destruction of alveolar walls in the lungs of mice exposed to CS. Original magnification: x400. The values of the mean linear intercept were increased significantly in CS-exposed mice. (B) Total RNA recovered from BAL cells was assayed using RT-PCR. Enhanced MafB gene expressions were observed in 6-mo CS-exposed (emphysema) mice compared with controls. (C) Immunostaining of MafB in BAL cells from emphysema mice and control mice. Intense staining of MafB was observed in BAL cells from emphysema mice compared with control mice. Note that only AMs (arrowhead) were strongly positive for MafB, but that neutrophils (arrow) were negative. Counterstaining: nuclear fast red. **P < 0.01 versus control.

 
Time-Course of MafB Expression in the BAL Cells of CS-Exposed Mice
To evaluate the time-course change of MafB expression in BAL cells after CS exposure, its expression was examined by RT-PCR. Total cells in BAL fluids were increased in the 2-wk CS-exposed mice compared with control mice. In addition, the numbers of AMs and neutrophils were increased significantly in the 2-wk CS-exposed mice (Table 2). The expressions of MafB mRNA of CS-exposed mice were increased gradually in a time-dependent manner for up to 2 wk (Figure 2, closed circle; median value: Day 0, 1.00; Day 1, 1.07; Day 3, 3.10; Day 7, 5.01; Day 14, 6.94), while those of control mice remained unchanged (Figure 2, open circle; median value: Day 0, 1.11; Day 1, 1.00; Day 3, 1.00; Day 7, 1.03; Day14, 1.02). The levels of MafB expression in 2-wk CS-exposed mice were as high as those in 6-mo CS-exposed mice.


Figure 2
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  		Figure 2. MafB mRNA expression in BAL cells of CS-exposed mice. Expression of MafB mRNA in BAL cells. Total RNA from BAL cells of control (open circles) and CS-exposed mice (filled circles) was assayed using RT-PCR. MafB mRNA expression was increased in a time-dependent manner up to 2 wk of CS exposure. The levels of MafB expression in 2-wk CS-exposed mice were as high as those in 6-mo CS-exposed mice. n >= 4 at each time point; *P < 0.05 versus control.

 

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  		TABLE 2. COMPARISON OF THE BAL CELL CONCENTRATIONS BETWEEN CONTROL AND 2-WK CS-EXPOSED MICE

 
Activation of MafB in BAL Cells of CS-Exposed Mice
The DNA binding activity of MafB was determined in nuclear extracts from the 2-wk CS-exposed mice by EMSA (Figure 3). Increased DNA-nuclear protein complex was observed in the BAL cells of the 2-wk CS-exposed mice compared with the control mice. Binding of the nuclear extracts to the MARE motif was inhibited by coincubation with the excess of unlabeled MARE oligonucleotide. Furthermore, addition of anti-MafB antibody to the binding reaction caused the disappearance of DNA–nuclear protein complex (32), indicating that this complex consisted of MafB protein and MARE probe.


Figure 3
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  		Figure 3. MafB activation in CS-exposed AMs. MafB activities in BAL cells obtained from control mice and 2-wk CS-exposed (acute smoking) mice were assessed using EMSA. Enhanced DNA-binding activities for MARE were observed in the nuclear extracts from acutely CS-exposed mice compared with those from control mice. Specific competition with an excess of unlabeled (cold) MARE oligonucleotide eliminated the band of DNA–nuclear protein complex. MafB antibody also eliminated the band of DNA–nuclear protein complex. A representative image from three experiments is shown.

 
Increased Oxidative Stress in Lungs of CS-Exposed Mice
We measured the levels of LPO in the lungs of control and 2-wk CS-exposed mice to estimate the burden of oxidative stress in the CS-exposed lung. Concentrations of LPO in the lung homogenates were significantly increased in the 2-wk CS-exposed mice compared with those of the control mice (Figure 4A; median value: control, 67.6 pmol/mg lung protein; smoking, 176.0 pmol/mg lung protein).


Figure 4
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  		Figure 4. 4-HNE–stimulated MafB gene expression in a murine macrophage cell line RAW264.7 cell. (A) After respective lung tissues from control mice and 2-wk CS-exposed mice (acutely CS-exposed mice) were homogenized, their LPO concentrations were determined. Concentrations of LPO were significantly higher in the lungs of 2-wk CS-exposed mice. (B) MafB gene expressions were assessed using RT-PCR in RAW264.7 cells treated with 20 µM 4-HNE for 18 h and cells untreated (control). (C) The reporter plasmid, which contained three MARE motif in the upstream of beta-lactamase cDNA was stably transfected into RAW cells (RAW264.7-pMARE-Bla cell). beta-lactamase activities were assessed using spectrofluorometry in RAW264.7-pMARE-Bla cells treated with 20 µM 4-HNE for 18 h and cells untreated (control). Results show that 4-HNE significantly activated MARE-mediated reporter gene expression. **P < 0.01, ***P < 0.001 versus control.

 
MafB Expression Was Increased by 4-HNE in Murine Macrophage Cell Line
The Maf family proteins are involved in multiple stress response, including oxidative stress (33, 34). To determine whether oxidative stress promotes MafB gene expression, murine macrophage cell line RAW264.7 cells were treated with 20 µM 4-HNE, a major product of lipid peroxidation (35). As expected, MafB expression was enhanced significantly by 4-HNE (Figure 4B; median value: control, 0.93; 4-HNE, 1.89). No toxicity was observed under this concentration of 4-HNE (trypan blue exclusion test, data not shown).

The elevated MafB activity by 4-HNE was confirmed using beta-lactamase reporter assay. By 4-HNE, the macrophage cell line stably transfected with the reporter plasmid, RAW264.7-pMARE-Bla cells, exhibited more intense beta-lactamase expression than that from untreated cells (Figure 4C; median value: control, 0.88; 4-HNE, 3.00).

Establishment of MafB-Overexpressing Macrophage Cell Line
We established MafB-overexpressing cell lines, RAW264.7-pcMafB, to investigate the role of MafB in macrophages. Figure 5A shows the overexpression of MafB protein in two selected clones, named as RAW264.7-pcMafB-#15 and RAW264.7-pcMafB-#20, in comparison with control cells (RAW264.7-pcCTRL). The activity of MafB in RAW264.7-pcMafB was confirmed through cotransfection of MARE-reporter plasmid (pMARE-Bla). The MafB-overexpressing cells, RAW264.7-pMARE-Bla-pcMafB, exhibited stronger beta-lactamase expression than did control cells, RAW264.7-pMARE-Bla-pcCTRL (Figure 5B; median value: pcCTRL, 1.13; pcMafB, 2.07).


Figure 5
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  		Figure 5. Establishment of MafB-overexpressing cells. (A) After RAW264.7 cells were transfected with control vector (pcCTRL) (left lanes) or murine MafB encoding vector (pcMafB) (right two lanes), MafB expression was evaluated using immunoblotting in each clone. The figure shows the expression of MafB in the representative MafB-overexpressing cells (RAW264.7-pcMafB-#15 and -#20). (B) Enhanced MafB activity in the MafB-overexpressing cells was confirmed by reporter assay. We transfected pcMafB or pcCTRL into RAW264.7-pMARE-Bla cells. beta-lactamase activities in the cell lysates were compared between RAW264.7-pMARE-Bla-pcMafB and RAW264.7-pMARE-Bla-pcCTRL cells using a spectrofluorometer. Overexpression of MafB significantly enhanced MARE-mediated reporter gene expression. **P < 0.01 versus control.

 
Enhanced Viability and Suppression of Apoptosis by MafB Overexpression in the Macrophage Cell Line
The prolonged survival of AMs is a notable aspect of smokers' lungs (6–8). Therefore, we investigated whether AM viability and apoptosis were changed by MafB overexpression in the macrophage cell line. Figure 6A shows that the cell viability after CSE treatment was enhanced significantly by MafB overexpression (median value: pcCTRL, 1.01; pcMafB, 1.53; P < 0.01). Next, we investigated the occurrence of apoptosis after CSE treatment by measuring the disintegrity of cell membranes (27). As shown in Figure 6B, MafB overexpression significantly inhibited apoptosis (median value: pcCTRL, 1.09; pcMafB, 0.65; P < 0.01). In addition, TUNEL-positive cells were decreased in MafB-overexpressing cells (Figure 6C).


Figure 6
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  		Figure 6. Overexpression of MafB attenuated CS-induced apoptosis. (A) RAW264.7-pcMafB cells and RAW264.7-pcCTRL cells were treated with 20% CSE for 12 h. Subsequently, their viability was evaluated using MTS assay. MafB-overexpressing cells had higher viability after CSE treatment than did control cells. (B) Occurrence of apoptosis after CSE-treatment was evaluated by APOPercentage Apoptosis Assay Kit. RAW264.7-pcMafB cells and RAW264.7-pcCTRL cells were treated with 20% CSE for 6 h. Subsequently, the apoptosing cells were stained with the dye according to the manufacturer's protocol. The absorbance at 550 nm in cell lysate was measured to quantify the apoptosing cells. MafB overexpression significantly suppressed apoptosis induced by CSE treatment. (C) Occurrence of apoptosis after CSE treatment was evaluated through TUNEL staining. RAW264.7-pcMafB cells and RAW264.7-pcCTRL cells were treated with 20% CSE for 6 h. Subsequently, TUNEL staining was performed. Fewer TUNEL-positive cells were observed in MafB-overexpressing cells (right panel) than in control cells (left panel). Microscopic images show representative fields. **P < 0.01 versus control.

 
We evaluated caspase activities in cells treated with CS to assess the mechanism involved in the inhibition of apoptosis by MafB overexpression. Figure 7A shows that only caspase-3–specific inhibitor, Z-DEVD-FMK, inhibited the occurrence of apoptosis induced by CSE in RAW264.7 cells (median value: untreated, 0.92; 20% CSE alone, 1.59; +Z-YVAD-FMK, 1.65; +Z-DEVD-FMK, 1.29; +Z-VEID-FMK, 1.64; +Z-IETD-FMK, 1.54; and +Z-LEHD-FMK, 1.61). Consistent with this finding, caspase-3 activity was up-regulated by CSE in a dose-dependent manner in the macrophage cell line (Figure 7B; median value: 0%, 0.98; 10%, 1.40; and 20%, 2.33). These findings suggest that caspase-3 might play an important role for CS-induced apoptosis in macrophages. Furthermore, caspase-3 activity was suppressed significantly by MafB overexpression (Figure 7C; median value: pcCTRL, 1.01; pcMafB, 0.66; P < 0.01).


Figure 7
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  		Figure 7. Overexpression of MafB inhibited caspase-3 activation after CSE-exposure. (A) Caspase-3 inhibitor suppressed occurrence of apoptosis induced by CSE-exposure. RAW264.7 cells were pretreated with 2 µM of caspase inhibitors for 3 h. Subsequently, 20% CSE was added. Occurrence of apoptosis was evaluated by APOPercentage Apoptosis Assay Kit 16 h later. Only caspase-3 inhibitor reduced the occurrence of apoptosis induced by CS. Z-YVAD-FMK, caspase-1 inhibitor; Z-DEVD-FMK, caspase-3 inhibitor; Z-VEID-FMK, caspase-6 inhibitor; Z-IETD-FMK, caspase-8 inhibitor; Z-LEHD-FMK, caspase-9 inhibitor. n = 6 in each group. (B) Activation of caspase-3 by CSE-treatment in the macrophage cell line. RAW 264.7 cells were treated with 0, 10, or 20% CSE for 16 h. Caspase-3 activity was enhanced by CSE in a dose-dependent manner. n = 6 in each group. (C) Inhibition of caspase-3 in MafB-overexpressing cells. Caspase-3 activity was examined in control and MafB-overexpressing cells after 20% CSE-treatment for 16 h. Caspase-3 activity was inhibited significantly by MafB overexpression. (D) Cytochrome c release by CSE-treatment. The release of cytochrome c from mitochondria into cytoplasm was assessed. Cells were treated with 0% or 20% of CSE for 16 h; thereafter, proteins of cytoplasmic (upper panel) and mitochondrial fractions (lower panel) were extracted, and the levels of cytochrome c were assessed by immunoblotting. The release of cytochrome c by CSE-treatment was not different between MafB-overexpressing and control cells. A representative image from three experiments is indicated. (E) RT-PCR analysis of apoptosis-related molecules in MafB overexpressing cells. Representative images of caspase-3, Apaf-1, Moap1, p21CIP/WAF1, BclXL, Bax, and GAPDH gene expressions from six experiments were indicated. Caspase-3 gene expression remained unchanged by MafB overexpression. Moap1 expression was attenuated significantly by MafB. *P < 0.05, **P < 0.01.

 
In apoptosing cells, mitochondrial cytochrome c is released into the cytoplasm (5, 36). After CSE treatment, the level of cytochrome c in cytoplasm was elevated both in MafB-overexpressing and control cells (Figure 7D, upper panel). Nevertheless, no differences of cytoplasmic cytochrome c levels are apparent between MafB-overexpressing and control cells (Figure 7D, upper panel). Moreover, the level of cytochrome c in mitochondria was not changed by CSE treatment in either cell line (Figure 7D, lower panel).

The MafB is a TF that has the potential to modulate expressions of various genes. However, caspase-3 expression was not altered by MafB overexpression (Figure 7E). Reportedly, caspase-3 activity is mediated by various molecules such as apoptotic peptidase–activated factor 1 (Apaf1) and modulator of apoptosis 1 (Moap1) (36, 37). In addition, p21CIP1/WAF1, BclXL, and Bax are also thought to be important molecules for CS-induced apoptosis in macrophages (5, 7). Figure 7E shows that Moap1 gene expression was reduced significantly by MafB overexpression (median value: pcCTRL, 0.46; pcMafB, 0.14; P < 0.05), whereas other gene expressions remained unchanged.

Overexpression of MafB Did Not Enhance the Replication Rate of Macrophages
We evaluated the difference of the proliferation rates of MafB-overexpressing and control cells. The proliferation rates of both cell lines were similar (Figure 8).


Figure 8
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  		Figure 8. Overexpression of MafB did not enhance the macrophage proliferation rate. Growth rate differences were assessed between MafB-overexpressing and control cells using MTS assay. Viable cell numbers at 0, 24, and 48 h were indicated as the ratio of the absorbance value against the mean absorbance value of each cell line at 0 h. Filled triangle, RAW264.7-pcCTRL cell line; filled circle, RAW264.7-pcMafB cell line. n = 6 at each time point in each group.

 

   DISCUSSION
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
Cigarette smoke is an important risk factor for development of many lung diseases, including COPD. Using an animal model of CS exposure, we investigated the expression of TFs that regulate the functions of macrophages. Among them, MafB mRNA expression was increased markedly (Figure 1B). As shown in Figure 3, increased DNA binding activity of MafB was confirmed in nuclear extracts from mice exposed to CS for 2 wk by EMSA. Addition of anti-MafB antibody to the binding reaction caused the disappearance of the DNA–nuclear protein complex. Usually, the addition of specific antibody causes supershift of the DNA–protein complex. However, it has also been known that addition of specific antibody can result in disappearance of its band instead of supershift (32). Although we did not confirm the activity of MafB in CS-exposed mice at 6 mo of CS exposure by EMSA, it was strongly suggested that MafB in 6-mo CS-exposed mice could be activated through the up-regulation of MafB mRNA and protein (Figures 1 and 2). To date, the expression and role of MafB in normal or diseased lungs has not been elucidated. In this article, we demonstrated that MafB is up-regulated in AMs of mice with emphysema induced by CS exposure (Figure 2), and showed its potential role for inhibition of apoptosis in macrophages.

To evaluate a role of MafB in the macrophages of smokers, 2 wk of CS exposure may have additional utility as surrogate for 6-mo CS exposure in mice. Although the duration of exposure we used may be too short to know the entire course of the development of emphysema, the animal model of 2-wk CS exposure may still useful to clarify a role of MafB for macrophage survival because (1) the level of MafB by CS exposure was as high as those in 6-mo CS-exposed mice (Figure 2), and (2) AMs were already increased in number by 2 wk of CS exposure.

CS contains more than 4,700 chemical compounds and oxidants. Small Maf proteins were induced by hydrogen peroxide (33) and heavy metals (34). Therefore, we hypothesized that MafB might be induced by oxidative stress. Initially, we confirmed that the lungs of CS-exposed mice were in an oxidant-rich environment by measuring LPO concentrations (Figure 4A). A specific end-product of LPO, 4-HNE, is a diffusible agent that can attack targets far from the original site of free radical generation (35). It has been reported that the level of 4-HNE was elevated in the lungs of patients with COPD (38) and mice acutely exposed to CS (39). These findings suggest that CS exposure induces LPO-related oxidative stress in the lung in the acute and chronic phases of cigarette smoking. Consistent with our hypothesis, 4-HNE induced expression of MafB (Figure 4B) and enhanced the transcriptional activity of MafB through MARE motif in a murine macrophage cell line (Figure 4C). The present data support the concept that LPO-related oxidative stress is associated with MafB gene expression in the CS-exposed lung.

To clarify the role of MafB in AMs, we established MafB-overexpressing macrophage cell lines. Because MafB is a proto-oncogene, MafB offers the potential to accelerate the cell cycle of macrophages (14, 40). In our observation, the growth rates of MafB-overexpressing and control cells were similar (Figure 8). However, control cells used in this study (RAW264.7 cell) is an Abelson leukemia virus–transformed cell line, and its growth rate probably differs from that of primary AMs. The replication rate of RAW264.7 cells might already reach a maximum. According to the evidence that MafB is a responsible protooncogene for multiple myeloma, MafB might induce cell proliferation in primary AMs (14, 15).

Many protooncogenes are known to enhance cell viability by inhibiting apoptosis (16). Thus, we investigated the effect of MafB on cellular viability and apoptosis after CSE treatment. Interestingly, MafB maintained the viability of macrophage cell line after CSE treatment. Consistent with this finding, apoptosis that was induced by CSE was inhibited significantly by MafB overexpression (Figure 6). Programmed cell death is observed in various cells in patients with COPD, and influences the viability of cells in the lung (5, 41, 42). These findings support the idea that MafB plays an important role for maintaining cell viability by inhibiting apoptosis in the AMs from lungs of smokers. Enhanced cell survival and increased AMs in smokers might be attributable to the inhibition of apoptosis by MafB. Nevertheless, a question still exists whether MafB inhibits the apoptosis of primary AMs in smokers. To address this question, fluorescence-activated cell sorting assay using MafB and Annexin V (as an apoptosis marker) antibodies is needed to confirm the inverse relationship between the expression of MafB and occurrence of apoptosis in primary AMs. Unfortunately, the MafB antibodies that we used in this study have not worked for the fluorescence-activated cell sorting assay. Thus, studies using gene-targeted animals are required to address this question in the future.

It was reported that CS-induced apoptosis of rat primary AMs is independent of any major caspases (5). In contrast, our data imply that CS-induced apoptosis of macrophage depends partly on caspase-3. Although the reason for this difference is unknown, the involvement of caspase-3 in CS-induced apoptosis was supported by two findings: (1) inhibitor of caspase-3 (Z-DEVD-FMK) reduced apoptosis by CSE (Figure 7A); and (2) caspase-3 was activated by CSE in the macrophage cell line in a dose-dependent manner (Figure 7B). Importantly, MafB overexpression significantly inhibited the caspase-3 activity (Figure 7C) and reduced apoptosis after CSE treatment (Figures 6B and 6C). Therefore, we propose that the inhibition of caspase-3 activity by MafB is an important mechanism for prolonged macrophage survival against CS-induced cytotoxicity.

Caspase activity is modulated by various caspase-related molecules, such as Moap1 and Apaf1. Because caspase-3 gene expression in MafB-overexpressing cells was not changed (Figure 7E), the activity of caspase-3 might be suppressed through alteration of caspase-related molecules by MafB. Among the molecules investigated, Moap1 was the only molecule that was repressed by MafB overexpression (Figure 7E). Moap1 is a novel proapoptotic protein containing a BH3-like motif that associates with Bax through its Bcl-2 homologous domains; it mediates caspase-dependent apoptosis in mammalian cells when overexpressed (37). Therefore, Moap1 is a candidate gene to regulate caspase-3 activity in CS-induced apoptosis.

Mitochondrial dysfunction is another important process for apoptosis (36). It has been reported that CS-induced apoptosis in rat primary AMs depends on mitochondrial dysfunction caused by up-regulation of Bax, a proapoptotic modulator (5). However, by CSE treatment, the release of cytochrome c in MafB-overexpressing cells was not different from that in control cells (Figure 7D). In addition, the expression of Bax and BclXL, which might be related to cytochrome c release in apoptosing cells, was not altered by MafB (Figure 7E). In this study, we observed no protective effect of MafB on mitochondrial functions in CS-exposed cells.

Small Maf proteins are important for expression of antioxidant and detoxification enzymes (43, 44). Small Maf family proteins dimerize with Nrf-2; they induce HO-1 and GSTP1, thereby protecting the cells from oxidative stress (33, 34, 45–49). In our study, HO-1 mRNA expression was slightly decreased (0.82- ± 0.11-fold increase versus control, P < 0.05, n = 6 in each group), and GSTP1 mRNA expression was not altered (0.96- ± 0.04-fold increase versus control, P = 0.6, n = 6 in each group) in the MafB-overexpressing cell lines. Thus, in contrast to small Maf proteins, MafB up-regulated neither HO-1 nor GSTP1 expression, indicating that MafB does not dimerize with Nrf-2 (50). Therefore, enhanced viability by MafB is unlikely to be caused by induction of antioxidant or detoxification enzymes.

In conclusion, we demonstrated a novel function of MafB in macrophages of smokers' lungs. Our results suggest that MafB expression in AMs is increased by CS exposure, and that enhanced expression of MafB might prevent AMs from apoptosis through inhibiting caspase-3 activity, resulting in survival prolongation of AMs. The surviving AMs from apoptosis might modulate the pathogenesis of CS-related lung diseases, including COPD.


   Acknowledgments
 
The authors thank Shuku Takahashi, Sachi Adachi, Miki Takase-Sato, and Eiji Tsuchida for their excellent technical assistance, and also thank Dr. John C. Gomez (Case Western Reserve University, Rainbow Babies and Children's Hospital) for the revision of language in this manuscript.


   Footnotes
 
This study was supported by a grant-in-aid from the 21st century center of excellence program of the Japan Society for the Promotion of Science, and grants-in-aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan (147700268, 15790407, 16590733, 17590778, 18590835, and 18790530).

Originally Published in Press as DOI: 10.1165/rcmb.2006-0248OC on November 1, 2006

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form July 11, 2006

Accepted in final form October 23, 2006


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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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