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EBPδ in Lung Epithelial Cells
Molecular Cell Biology Research Communications 3, 42– 47 (2000) doi:10.1006/mcbr.2000.0187, available online at http://www.idealibrary.com on
CYP2B1 Is Regulated by C/EBP␣ and C/EBP␦ in Lung Epithelial Cells Tobias N. Cassel, Jan-Åke Gustafsson, and Magnus Nord 1 Department of Medical Nutrition, Karolinska Institutet, Huddinge University Hospital, F60 Novum, S-141 86 Huddinge, Sweden
Received January 3, 2000
CYP2B1 has been demonstrated in the nonciliated Clara cells of the proximal and distal airways as well as lower levels in the type II cells of the alveolar region (2–5). In contrast, in the liver, CYP2B1 expression is low under normal conditions but is induced after phenobarbital treatment (6). In rat lung, CYP2B1 expression is first detected from day 4 postnatal age and adult levels are reached 4 – 6 weeks after birth (7, 8). The factors involved in the differentiation-dependent expression of CYP2B1 in the lung are largely unknown. In a recent study it was demonstrated that ⫺1.3 kb of the 5⬘ flanking region of the CYP2B1 promoter is sufficient to give lung-specific expression of this gene (9). In liver cells it has been demonstrated that important positive cis-acting elements reside in the proximal promoter of CYP2B genes (10, 11). It was demonstrated that the CYP2B1 gene is transcriptionally activated through one of these cisacting elements by the C/EBP␣ and C/EBP in HepG2 cells of liver origin (11). Deletion or mutation of this proximal C/EBP-binding site resulted in markedly reduced promoter activity (10, 11). However, this C/EBPbinding was not responsive to phenobarbital and its mutation did not affect phenobarbital-enhanced reporter gene activity (10, 12). C/EBP factors are known to play important roles in controlling differentiation and differentiation-dependent processes in several organs, most notably in liver and fat. In these organs, C/EBP factors are important regulators of proliferation, cell cycle arrest and gene expression (13–15). In rat lung, C/EBP␣ is expressed in a differentiationdependent manner and is first detected from embryonic day 17–18, in close correlation to the onset of Clara cell differentiation (19). Apart from C/EBP␣, C/EBP␦ is expressed at high levels in the lung (20). Although the expression of C/EBP-factors in the lung is well known, few studies have investigated the involvement of these transcription factors in lung-specific gene expression. In the present study, we demonstrate a role for C/EBP␣ and C/EBP␦ in CYP2B1 expression in lung epithelial cells and thus a putative role for
Pulmonary expression of several cytochrome P450 (CYP) monooxygenases is detected late in gestation. Little is known of the factors involved in this differentiation-dependent expression. C/EBP factors are known regulators of differentiation and differentiation-dependent gene expression in several tissues. In this study we demonstrate the importance of C/EBP␣ and C/EBP␦ in pulmonary epithelial CYP2B1 gene expression. A 1.3 kb CYP2B1 promoter fragment which recently has been shown to confer lung tissue- and cell-specific expression of CYP2B1 in transgenic mice was used in transient transfection studies. Both C/EBP␣ and C/EBP␦ transactivated the CYP2B1 promoter in the lung epithelial cell lines A549 and NCI-H441. C/EBP␣ in nuclear extracts from isolated rat primary bronchiolar Clara cells was capable of interacting with a C/EBP-binding site in the proximal CYP2B1 promoter. Site-directed mutagenesis studies showed that this proximal C/EBP-binding site is necessary for transactivation of the CYP2B1 gene by C/EBP␣ and C/EBP␦. This study shows that C/EBP factors have a role in pulmonary CYP2B1 expression and suggests that these transcription factors may be important for the differentiation-dependent expression of CYP2B1 in the lung. © 2000 Academic Press
Cytochromes P450 (CYP) constitute a superfamily which consists of several classes of membrane bound hemoproteins known to be involved in catalyzing the metabolism of a wide variety of substrates. The expression of these proteins is localized mainly to organs such as the kidney, brain, liver, and lung. In rat lung, the dominant P450 enzyme is the CYP2B1 isoform (1). The lung is a major target organ for many environmental pollutants and high-level constitutive expression of Abbreviations used: CYP, cytochrome P450; CCSP, Clara cell secretory protein; C/EBP, CCAAT/enhancer binding protein; CMV, cytomegalovirus; EMSA, electrophoretic mobility shift assay. 1 To whom correspondence should be addressed. Fax: ⫹46-8-711 6659. E-mail: [email protected]. 1522-4724/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
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these transcription factors in the differentiationdependent expression of this enzyme.
Electrophoretic Mobility Shift Assay (EMSA) An oligonucleotide spanning the previously characterized C/EBP-binding site in the CYP2B1 promoter (11) was synthesized (CAT CTG AAG TTG CAT AAC TGA G). EMSAs were performed by incubating 2 ⫻ 10 6 cpm of end-labeled oligonucleotide probe together with nuclear protein (1– 8 g) in 20 l of binding buffer (20 mM Hepes, pH 7.6, 40 mM KCl, 2 mM MgCl 2, 1 mM dithiothreitol, 0.5 mM EGTA, 4% Ficoll and 2 g of poly(dI:dC) as nonspecific competitor) for 15 min at room temperature. Before addition of labeled probe, samples were preincubated in binding buffer for 15 min at room temperature. In some experiments, excess of unlabeled double-stranded oligonucleotide or 1 l of polyclonal antisera were added in the preincubation step. Polyclonal antibodies to C/EBP␣ and C/EBP␦ were from Santa Cruz Biotechnology (CA). Protein– DNA complexes were resolved on a preelectrophoresed, nondenaturing 5% polyacrylamide gel with 0.5⫻ TBE as running buffer. Gels were vacuum-dried and exposed to autoradiographic film.
MATERIALS AND METHODS Cell Culture and Transfections The continuous cell lines A549 and COS-1 were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum, penicillin (100 IU/ml), and streptomycin (0.1 mg/ml). NCI-H441 cells were cultured in RPMI 1640 supplemented as above. For transient transfection experiments, A549 and H441 cells were seeded in 30-mm-diameter cell culture wells 24 h before a transfection experiment and transfected at approximately 60% confluence. Plasmid DNAs were transfected into the cells by liposome mediated DNA transfer using the LipofectAMINE transfection reagent (GIBCO-BRL, UK), according to the protocol provided by the manufacturer. 2.4 g of the reporter plasmid p2B1, including 1350 bp of the proximal CYP2B1 in front of a -galactosidase reporter gene (9; a kind gift from Rune Toftgård, Dept. of Biosciences, NOVUM, Karolinska Institute, Sweden), was used in each transfection experiment together with 0.01 or 0.05 g of pCMVC/EBP expression plasmid. In some experiments, transfection of 0.01 g was excluded. To normalize for variations in transfection efficiency, a plasmid containing a luciferase reporter gene driven by an RSV promoter was included in each experiment. Reporter gene activity was measured using the Galacto Light Plus -galactosidase assay kit (Tropix, MA) and the Genglow 1000 luciferase assay kit (BioOrbit, Finland) on an Anthos Lucy 1 luminometer (Anthos Labtech Instruments, Austria) for -galactosidase and luciferase activity, respectively. For overexpression of C/EBP-factors, COS-1 cells were transiently transfected using the DEAE-dextran method (16). Briefly, the cells were washed with serum-free DMEM medium before a two hour incubation in serum-free DMEM transfection medium, with 20 g murine C/EBP␣ or C/EBP␦ expression plasmid and 500 g DEAE dextran. Cells were harvested 72 h after transfection and nuclear proteins were prepared as described previously (17).
SDS–Polyacrylamide Gel Electrophoresis and Western Immunoblotting For detection of C/EBP␦, 45 g of nuclear proteins from primary Clara cells and 10 g nuclear proteins from COS cells transfected with a C/EBP␦ expression vector were separated on a 12% SDS–PAGE and transferred to a nitrocellulose filter by electroblotting. The filter was blocked in 5% nonfat dry milk in 10 mM Tris pH 7.4, 150 mM NaCl, 0.1% Tween (TBS/Tween), for 1 hour at room temperature, washed with TBS/Tween and subsequently incubated with primary C/EBP␦ antibody (Santa Cruz Biotechnology, CA) at 4°C over night. Bound antibody was visualized by the enhanced chemiluminescence system (ECL; Amersham Pharmacia Biotech, UK) using a goat anti-rabbit peroxidaseconjugated secondary antibody. Site-Directed Mutagenesis The plasmid p2B1, including 1350 bp of the CYP2B1 promoter (9), served as template for sitedirected mutagenesis using the QuikChange sitedirected mutagenesis kit (Stratagene, CA). All reactions were performed according to the protocol provided by the manufacturer. The oligonucleotides used for site-directed mutagenesis of the C/EBPbinding site were as follows (complementary strands are not shown, mutated bases are underlined): GAA CAT GTG AAG TTG CAT CGC TGA GTG GAG GGG. To verify the correct mutation, the construct was subjected to DNA sequence analysis.
Isolation of Primary Rat Clara Cells Isolation of primary rat Clara cells was carried out as previously described (4). Briefly, lungs were perfused with 37°C buffer before removal. Subsequently, the lungs were digested with protease, minced and filtered to give a single-cell suspension. Cells were then separated by size via centrifugal elutriation. This was followed by incubation of the cell suspension in regular petri dishes to allow nonepithelial cells to attach. From the purified Clara cell fraction, nuclear proteins were prepared as described previously (17). 43
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RESULTS C/EBP␣ and C/EBP␦ Transactivate the CYP2B1 Gene in Lung Epithelial Cells To determine whether the rat CYP2B1 gene is directly regulated by C/EBP␣ in lung epithelial cells, the effects of this transcription factor on CYP2B1 transcription were examined by transient transfection assays. For these studies, a -galactosidase reporter plasmid containing a 1350 bp promoter region of the rat CYP2B1 gene was used. This part of the CYP2B1 promoter has recently been shown in a transgenic mouse model to confer tissue and cell-specific expression of a reporter gene reflecting the endogenous CYP2B1 expression in the lung (9). The continuous lung epithelial cell line A549 was transiently transfected with the CYP2B1-promoter construct together with increasing amounts of C/EBP␣ expression plasmid. This resulted in a maximal induction of reporter gene activity reaching 30-fold (Figure 1A). The expression of another C/EBP family member, C/EBP␦, parallels the expression pattern of CYP2B1 in that C/EBP␦ is constitutively expressed at high levels in the lung, with the highest levels seen in the Clara cells and lower levels in cells of the alveolar region (18). Thus, further transfections using a C/EBP␦ expression plasmid were performed. When increasing amounts of C/EBP␦ expression plasmid were transfected to A549 cells, the observed maximal induction of CYP2B1 promoter activity was 53-fold (Figure 1A). The A549 cell line is generally considered to be of alveolar type II cellorigin. As CYP2B1 is expressed in the bronchiolar Clara cells as well (2–5) the transfection studies were expanded into the Clara cell-like NCI-H441 lung epithelial cell line. When increasing amounts of C/EBP␣ or C/EBP␦ expression plasmids were transfected into H441 cells, the induction of CYP2B1 promoter activity was 10-fold and 60-fold by C/EBP␣ and C/EBP␦, respectively (Figure 1B). The lower activity of C/EBP␣ in H441 cells compared to A549 cells is probably reflecting endogenous C/EBP␣ expression in H441 cells (19).
FIG. 1. Cotransfection studies to determine transactivation of the CYP2B1 promoter by C/EBP␣ and C/EBP␦. The reporter plasmid contained a 1350 bp fragment of the 5⬘ flanking region of the CYP2B1 promoter. Increasing amounts of pCMVC/EBP␣ or pCMVC/EBP␦ expression plasmids were cotransfected in (A) A549 lung epithelial cells or (B) H441 lung epithelial cells with constant amounts of reporter plasmid. Transfection efficiencies were normalized by cotransfection of a luciferase reporter plasmid. Error bars represent SEM (n ⫽ 6).
C/EBP␣ in Isolated Primary Rat Clara Cell Nuclear Extract Interacts with the Proximal CYP2B1 Promoter
abolished by competition with excess of unlabeled homologous oligonucleotide. Inclusion of C/EBP␣ antibody resulted in a supershift indicating binding of this factor to the C/EBP-binding site in primary Clara cells. Although C/EBP␦ is highly expressed in murine lung (20) and specifically in Clara cells (18), inclusion of C/EBP␦ antibody in the EMSA reaction did not alter the DNA-protein interaction pattern. To investigate the presence of C/EBP␦ in the rat primary Clara cell nuclear extracts, Western blotting was performed. In the Clara cell nuclear extract, a C/EBP␦ immunoreactive band of the expected size was detected (Figure 2B). However, this band was slightly different in size compared to overexpressed C/EBP␦ from COS cells (Figure
Previously, it has been demonstrated that C/EBP factors interact with a C/EBP-binding site in the proximal CYP2B1 promoter in the liver hepatoma cell line HepG2 (11). To determine the interaction of C/EBP factors with the CYP2B1 promoter in lung epithelial cells, an oligonucleotide including this previously characterized C/EBP-binding site was synthesized. In electrophoretic mobility shift assays (EMSAs), incubating this oligonucleotide together with nuclear extract from primary Clara cells isolated from rat, several shifts appeared (Figure 2A). These bands were efficiently 44
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FIG. 2. C/EBP-binding to the CYP2B1 promoter. (A) Electrophoretic mobility shift assay using nuclear extract from primary rat Clara cells incubated with an end-labeled oligonucleotide spanning the C/EBP-binding site in the CYP2B1 promoter (lane 1) and competed with a 200-fold excess of unlabeled homologous oligonucleotide (lane 2). In the presence of C/EBP␣ antibody, a super-shift appeared (lane 3) while inclusion of C/EBP␦ antibody did not alter DNA–protein interaction pattern (lane 4). (B) Western blot analysis for C/EBP␦ using Clara cell nuclear extract (lane 1) and nuclear extract from COS cells overexpressing C/EBP␦ (lane 2). Positions of molecular weight markers are indicated at left. (C) Labeled oligonucleotide was used for electrophoretic mobility shift assays with nuclear extracts from COS cells overexpressing C/EBP␣ (lane 4) or C/EBP␦ (lane 2) or C/EBP␦ together with anti-C/EBP␦ antibody (lane 3). As a control, nuclear extract from null-transfected COS cells was incubated with labeled oligonucleotide (lane 5). In lane 1, no nuclear extract was included. Indicated are specific retarded complex from rat Clara cells (I), super-shifted complex in the presence of antibody (ss), C/EBP␣ and ␦ containing complexes (␣ and ␦, respectively) and migration of free unbound oligonucleotide (free).
2B). To determine whether overexpressed C/EBP␦ was capable of interaction with the C/EBP-binding site in the CYP2B1 promoter, additional EMSAs were performed using COS cell nuclear extract. In the presence of C/EBP␣ or C/EBP␦ containing nuclear extracts, prominent DNA-protein interactions were demonstrated (Figure 2C). The C/EBP␦ shift could be supershifted upon inclusion of C/EBP␦ antibody.
results in a major decrease of promoter activity in transient transfection studies, the phenobarbitalinduction of the promoter is not affected by inactivation of the site (10 –12). Together these results suggest that C/EBP factors are major contributors to basal CYP2B1 expression and are not important for the phenobarbital induction of CYP2B1 in the liver. In the
Inactivation of the C/EBP-Binding Site in the CYP2B1 Promoter Results in Markedly Reduced Transactivation of C/EBP␣ and C/EBP␦ To determine the functional importance of the characterized C/EBP-binding site in lung epithelial cells, site-directed mutagenesis studies were performed. In transient transfection studies using a construct including the ⫺1350 bp proximal CYP2B1 promoter with the C/EBP-binding site inactivated, a marked reduction of the transactivation efficiency was seen from both C/EBP␣ and C/EBP␦ (Figure 3). This indicates that this C/EBP-binding site is the major site conferring transactivation of the CYP2B1-gene by C/EBP transcription factors in lung epithelial cells. FIG. 3. Functional analysis of the C/EBP-binding site in CYP2B1 promoter by cotransfection studies using a promoterreporter gene construct altered by site-directed mutagenesis. The ⫺1350 CYP2B1 promoter--galactosidase construct was subjected to site-directed mutagenesis in order to alter the DNA sequence of the C/EBP-binding site. pCMVC/EBP␣ or pCMVC/EBP␦ expression plasmids were cotransfected in A549 lung cells with constant amounts of promoter-reporter gene construct with wild-type (wt) or mutated (mut) promoter sequence. Transfection efficiencies were normalized as in Figure 1. Error bars represent SEM (n ⫽ 6).
DISCUSSION In this study we demonstrate that C/EBP␣ and C/EBP␦ regulate the CYP2B1 gene through interaction with a proximal C/EBP-binding site in lung epithelial cells. This C/EBP-binding site has previously been characterized in liver cells (11). Although it has been demonstrated that mutation of this C/EBP-binding site 45
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lung, CYP2B1 is constitutively expressed in bronchiolar Clara cells and at lower levels in alveolar type II cells. C/EBP␣ expression has been demonstrated in the alveolar region and at lower levels in the bronchiolar epithelium (18, 19). In contrast, C/EBP␦ is expressed at high levels in bronchiolar Clara cells and at lower levels in the alveolar region (18). In this study, we demonstrate that C/EBP␦ exhibits a higher transactivation potential compared to C/EBP␣ of the CYP2B1 gene. A higher transactivation potential for C/EBP␦ was evident from the transfection studies in both the A549 cells and in the Clara cell-like NCI-H441 cells. However, it is possible that the lower transactivation potential seen for C/EBP␣ in H441 cells reflects endogenous C/EBP␣ expression. In line with these results, it is possible that the higher levels of C/EBP␦ detected in the bronchiolar epithelium might explain the higher levels of CYP2B1 seen in this region. Moreover, in the liver, the levels of C/EBP␦ are low under normal conditions (20) perhaps explaining the low levels of CYP2B1 seen in this organ under non-induced conditions. CYP2B1 is a major enzyme responsible for the activation of tobacco-specific carcinogens in lung cells (21). The demonstration of a role for C/EBP factors in the regulation of pulmonary expression of CYP2B1 may thus help us understand mechanisms affecting susceptibility of the lung to injury from tobacco smoke. In EMSAs, no interaction of C/EBP␦ with the C/EBPbinding site in the CYP2B1 promoter was detected in rat primary Clara cell nuclear extract. To exclude the possibility that the C/EBP␦ isoform was incapable of interaction with the C/EBP-binding site in the CYP2B1 promoter, EMSAs were performed using nuclear extract from COS cells overexpressing C/EBP␦. These experiments demonstrated a strong interaction of C/EBP␦ with the CYP2B1 promoter. The finding that no interaction of C/EBP␦ was seen in the primary Clara cell nuclear extracts was unexpected as this protein has been shown to be highly expressed in murine lung and in Clara cells specifically (18, 20). Moreover, in Western blotting experiments it was shown that C/EBP␦ was present in the rat Clara cell nuclear extract. Together these data suggest that C/EBP␦ is prevented from interaction with the CYP2B1 promoter in nuclear extract from primary Clara cells. Previously it has been demonstrated that C/EBP␦ is regulated in its DNA-binding properties by phosphorylation in that it is posttranslationally modified by a member of the signal-transducing protein kinase cascade (22). Thus, it is possible that C/EBP␦ is modified in a way that prevents DNA-binding in the primary Clara cells. Furthermore, a change in phosphorylation of C/EBP␦ could explain the small size difference that was observed in the Western blot analysis between C/EBP␦ originating from primary Clara cells and overexpressed C/EBP␦ from COS cells. However, it can not be excluded that dephosphorylation of C/EBP␦ occur dur-
ing the Clara cell isolation process and thus these results do not necessary reflect the in vivo situation. C/EBP factors play important roles in controlling differentiation and differentiation-dependent processes. In many tissues, most notably in liver and fat, C/EBP factors are important regulators of proliferation, cell cycle arrest and gene expression (15). In rat lung, C/EBP␣ is expressed in a differentiationdependent pattern and is first detected around embryonic day 17–18 (19). Thus, the expression of C/EBP␣ parallels the cellular differentiation of the lung which takes place late during development resulting in highly specialized cell types such as the Clara cells of the conducting airways and the alveolar type II cells. The differentiation of these cell types is accompanied by expression of differentiation markers such as surfactant proteins, the Clara cell secretory protein (CCSP) and different P450 enzymes. In rat lung, CYP2B1 expression is detected late in development, around day 4 postnatal age (7, 8). We have previously shown that C/EBP factors are important for the regulation of the differentiation-dependent gene CCSP (18, 23). The results in the present study, indicating a role for C/EBP␣ and C/EBP␦ in the expression of CYP2B1, suggest that C/EBP factors have a more general role in differentiation-dependent gene expression in the lung epithelium. ACKNOWLEDGMENTS We are grateful to Per E. Schwarze, Marit Låg, and their group for isolation of primary rat Clara cells. We thank Rune Toftgård for the kind gift of reagents and Lena Nordlund-Mo¨ller for expert technical assistance. This work was supported by the Swedish Medical Research Council (Grant 13115), Swedish Match (Grant 199831), the Swedish Medical Society, the Swedish Heart–Lung Foundation, the research foundations “Tore Nilssons stiftelse fo¨r medicinsk forskning,” “Stiftelsen Lars Hiertas minne,” “Stiftelsen Sigurd och Elsa Goljes minne,” “Stiftelsen cystisk fibros forskningsfond,” and the Research Foundations of the Karolinska Institute.
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CYP2B1 Is Regulated by C/EBP␣ and C/EBP␦ in Lung Epithelial Cells Tobias N. Cassel, Jan-Åke Gustafsson, and Magnus Nord 1 Department of Medical Nutrition, Karolinska Institutet, Huddinge University Hospital, F60 Novum, S-141 86 Huddinge, Sweden
Received January 3, 2000
CYP2B1 has been demonstrated in the nonciliated Clara cells of the proximal and distal airways as well as lower levels in the type II cells of the alveolar region (2–5). In contrast, in the liver, CYP2B1 expression is low under normal conditions but is induced after phenobarbital treatment (6). In rat lung, CYP2B1 expression is first detected from day 4 postnatal age and adult levels are reached 4 – 6 weeks after birth (7, 8). The factors involved in the differentiation-dependent expression of CYP2B1 in the lung are largely unknown. In a recent study it was demonstrated that ⫺1.3 kb of the 5⬘ flanking region of the CYP2B1 promoter is sufficient to give lung-specific expression of this gene (9). In liver cells it has been demonstrated that important positive cis-acting elements reside in the proximal promoter of CYP2B genes (10, 11). It was demonstrated that the CYP2B1 gene is transcriptionally activated through one of these cisacting elements by the C/EBP␣ and C/EBP in HepG2 cells of liver origin (11). Deletion or mutation of this proximal C/EBP-binding site resulted in markedly reduced promoter activity (10, 11). However, this C/EBPbinding was not responsive to phenobarbital and its mutation did not affect phenobarbital-enhanced reporter gene activity (10, 12). C/EBP factors are known to play important roles in controlling differentiation and differentiation-dependent processes in several organs, most notably in liver and fat. In these organs, C/EBP factors are important regulators of proliferation, cell cycle arrest and gene expression (13–15). In rat lung, C/EBP␣ is expressed in a differentiationdependent manner and is first detected from embryonic day 17–18, in close correlation to the onset of Clara cell differentiation (19). Apart from C/EBP␣, C/EBP␦ is expressed at high levels in the lung (20). Although the expression of C/EBP-factors in the lung is well known, few studies have investigated the involvement of these transcription factors in lung-specific gene expression. In the present study, we demonstrate a role for C/EBP␣ and C/EBP␦ in CYP2B1 expression in lung epithelial cells and thus a putative role for
Pulmonary expression of several cytochrome P450 (CYP) monooxygenases is detected late in gestation. Little is known of the factors involved in this differentiation-dependent expression. C/EBP factors are known regulators of differentiation and differentiation-dependent gene expression in several tissues. In this study we demonstrate the importance of C/EBP␣ and C/EBP␦ in pulmonary epithelial CYP2B1 gene expression. A 1.3 kb CYP2B1 promoter fragment which recently has been shown to confer lung tissue- and cell-specific expression of CYP2B1 in transgenic mice was used in transient transfection studies. Both C/EBP␣ and C/EBP␦ transactivated the CYP2B1 promoter in the lung epithelial cell lines A549 and NCI-H441. C/EBP␣ in nuclear extracts from isolated rat primary bronchiolar Clara cells was capable of interacting with a C/EBP-binding site in the proximal CYP2B1 promoter. Site-directed mutagenesis studies showed that this proximal C/EBP-binding site is necessary for transactivation of the CYP2B1 gene by C/EBP␣ and C/EBP␦. This study shows that C/EBP factors have a role in pulmonary CYP2B1 expression and suggests that these transcription factors may be important for the differentiation-dependent expression of CYP2B1 in the lung. © 2000 Academic Press
Cytochromes P450 (CYP) constitute a superfamily which consists of several classes of membrane bound hemoproteins known to be involved in catalyzing the metabolism of a wide variety of substrates. The expression of these proteins is localized mainly to organs such as the kidney, brain, liver, and lung. In rat lung, the dominant P450 enzyme is the CYP2B1 isoform (1). The lung is a major target organ for many environmental pollutants and high-level constitutive expression of Abbreviations used: CYP, cytochrome P450; CCSP, Clara cell secretory protein; C/EBP, CCAAT/enhancer binding protein; CMV, cytomegalovirus; EMSA, electrophoretic mobility shift assay. 1 To whom correspondence should be addressed. Fax: ⫹46-8-711 6659. E-mail: [email protected]. 1522-4724/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
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these transcription factors in the differentiationdependent expression of this enzyme.
Electrophoretic Mobility Shift Assay (EMSA) An oligonucleotide spanning the previously characterized C/EBP-binding site in the CYP2B1 promoter (11) was synthesized (CAT CTG AAG TTG CAT AAC TGA G). EMSAs were performed by incubating 2 ⫻ 10 6 cpm of end-labeled oligonucleotide probe together with nuclear protein (1– 8 g) in 20 l of binding buffer (20 mM Hepes, pH 7.6, 40 mM KCl, 2 mM MgCl 2, 1 mM dithiothreitol, 0.5 mM EGTA, 4% Ficoll and 2 g of poly(dI:dC) as nonspecific competitor) for 15 min at room temperature. Before addition of labeled probe, samples were preincubated in binding buffer for 15 min at room temperature. In some experiments, excess of unlabeled double-stranded oligonucleotide or 1 l of polyclonal antisera were added in the preincubation step. Polyclonal antibodies to C/EBP␣ and C/EBP␦ were from Santa Cruz Biotechnology (CA). Protein– DNA complexes were resolved on a preelectrophoresed, nondenaturing 5% polyacrylamide gel with 0.5⫻ TBE as running buffer. Gels were vacuum-dried and exposed to autoradiographic film.
MATERIALS AND METHODS Cell Culture and Transfections The continuous cell lines A549 and COS-1 were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum, penicillin (100 IU/ml), and streptomycin (0.1 mg/ml). NCI-H441 cells were cultured in RPMI 1640 supplemented as above. For transient transfection experiments, A549 and H441 cells were seeded in 30-mm-diameter cell culture wells 24 h before a transfection experiment and transfected at approximately 60% confluence. Plasmid DNAs were transfected into the cells by liposome mediated DNA transfer using the LipofectAMINE transfection reagent (GIBCO-BRL, UK), according to the protocol provided by the manufacturer. 2.4 g of the reporter plasmid p2B1, including 1350 bp of the proximal CYP2B1 in front of a -galactosidase reporter gene (9; a kind gift from Rune Toftgård, Dept. of Biosciences, NOVUM, Karolinska Institute, Sweden), was used in each transfection experiment together with 0.01 or 0.05 g of pCMVC/EBP expression plasmid. In some experiments, transfection of 0.01 g was excluded. To normalize for variations in transfection efficiency, a plasmid containing a luciferase reporter gene driven by an RSV promoter was included in each experiment. Reporter gene activity was measured using the Galacto Light Plus -galactosidase assay kit (Tropix, MA) and the Genglow 1000 luciferase assay kit (BioOrbit, Finland) on an Anthos Lucy 1 luminometer (Anthos Labtech Instruments, Austria) for -galactosidase and luciferase activity, respectively. For overexpression of C/EBP-factors, COS-1 cells were transiently transfected using the DEAE-dextran method (16). Briefly, the cells were washed with serum-free DMEM medium before a two hour incubation in serum-free DMEM transfection medium, with 20 g murine C/EBP␣ or C/EBP␦ expression plasmid and 500 g DEAE dextran. Cells were harvested 72 h after transfection and nuclear proteins were prepared as described previously (17).
SDS–Polyacrylamide Gel Electrophoresis and Western Immunoblotting For detection of C/EBP␦, 45 g of nuclear proteins from primary Clara cells and 10 g nuclear proteins from COS cells transfected with a C/EBP␦ expression vector were separated on a 12% SDS–PAGE and transferred to a nitrocellulose filter by electroblotting. The filter was blocked in 5% nonfat dry milk in 10 mM Tris pH 7.4, 150 mM NaCl, 0.1% Tween (TBS/Tween), for 1 hour at room temperature, washed with TBS/Tween and subsequently incubated with primary C/EBP␦ antibody (Santa Cruz Biotechnology, CA) at 4°C over night. Bound antibody was visualized by the enhanced chemiluminescence system (ECL; Amersham Pharmacia Biotech, UK) using a goat anti-rabbit peroxidaseconjugated secondary antibody. Site-Directed Mutagenesis The plasmid p2B1, including 1350 bp of the CYP2B1 promoter (9), served as template for sitedirected mutagenesis using the QuikChange sitedirected mutagenesis kit (Stratagene, CA). All reactions were performed according to the protocol provided by the manufacturer. The oligonucleotides used for site-directed mutagenesis of the C/EBPbinding site were as follows (complementary strands are not shown, mutated bases are underlined): GAA CAT GTG AAG TTG CAT CGC TGA GTG GAG GGG. To verify the correct mutation, the construct was subjected to DNA sequence analysis.
Isolation of Primary Rat Clara Cells Isolation of primary rat Clara cells was carried out as previously described (4). Briefly, lungs were perfused with 37°C buffer before removal. Subsequently, the lungs were digested with protease, minced and filtered to give a single-cell suspension. Cells were then separated by size via centrifugal elutriation. This was followed by incubation of the cell suspension in regular petri dishes to allow nonepithelial cells to attach. From the purified Clara cell fraction, nuclear proteins were prepared as described previously (17). 43
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RESULTS C/EBP␣ and C/EBP␦ Transactivate the CYP2B1 Gene in Lung Epithelial Cells To determine whether the rat CYP2B1 gene is directly regulated by C/EBP␣ in lung epithelial cells, the effects of this transcription factor on CYP2B1 transcription were examined by transient transfection assays. For these studies, a -galactosidase reporter plasmid containing a 1350 bp promoter region of the rat CYP2B1 gene was used. This part of the CYP2B1 promoter has recently been shown in a transgenic mouse model to confer tissue and cell-specific expression of a reporter gene reflecting the endogenous CYP2B1 expression in the lung (9). The continuous lung epithelial cell line A549 was transiently transfected with the CYP2B1-promoter construct together with increasing amounts of C/EBP␣ expression plasmid. This resulted in a maximal induction of reporter gene activity reaching 30-fold (Figure 1A). The expression of another C/EBP family member, C/EBP␦, parallels the expression pattern of CYP2B1 in that C/EBP␦ is constitutively expressed at high levels in the lung, with the highest levels seen in the Clara cells and lower levels in cells of the alveolar region (18). Thus, further transfections using a C/EBP␦ expression plasmid were performed. When increasing amounts of C/EBP␦ expression plasmid were transfected to A549 cells, the observed maximal induction of CYP2B1 promoter activity was 53-fold (Figure 1A). The A549 cell line is generally considered to be of alveolar type II cellorigin. As CYP2B1 is expressed in the bronchiolar Clara cells as well (2–5) the transfection studies were expanded into the Clara cell-like NCI-H441 lung epithelial cell line. When increasing amounts of C/EBP␣ or C/EBP␦ expression plasmids were transfected into H441 cells, the induction of CYP2B1 promoter activity was 10-fold and 60-fold by C/EBP␣ and C/EBP␦, respectively (Figure 1B). The lower activity of C/EBP␣ in H441 cells compared to A549 cells is probably reflecting endogenous C/EBP␣ expression in H441 cells (19).
FIG. 1. Cotransfection studies to determine transactivation of the CYP2B1 promoter by C/EBP␣ and C/EBP␦. The reporter plasmid contained a 1350 bp fragment of the 5⬘ flanking region of the CYP2B1 promoter. Increasing amounts of pCMVC/EBP␣ or pCMVC/EBP␦ expression plasmids were cotransfected in (A) A549 lung epithelial cells or (B) H441 lung epithelial cells with constant amounts of reporter plasmid. Transfection efficiencies were normalized by cotransfection of a luciferase reporter plasmid. Error bars represent SEM (n ⫽ 6).
C/EBP␣ in Isolated Primary Rat Clara Cell Nuclear Extract Interacts with the Proximal CYP2B1 Promoter
abolished by competition with excess of unlabeled homologous oligonucleotide. Inclusion of C/EBP␣ antibody resulted in a supershift indicating binding of this factor to the C/EBP-binding site in primary Clara cells. Although C/EBP␦ is highly expressed in murine lung (20) and specifically in Clara cells (18), inclusion of C/EBP␦ antibody in the EMSA reaction did not alter the DNA-protein interaction pattern. To investigate the presence of C/EBP␦ in the rat primary Clara cell nuclear extracts, Western blotting was performed. In the Clara cell nuclear extract, a C/EBP␦ immunoreactive band of the expected size was detected (Figure 2B). However, this band was slightly different in size compared to overexpressed C/EBP␦ from COS cells (Figure
Previously, it has been demonstrated that C/EBP factors interact with a C/EBP-binding site in the proximal CYP2B1 promoter in the liver hepatoma cell line HepG2 (11). To determine the interaction of C/EBP factors with the CYP2B1 promoter in lung epithelial cells, an oligonucleotide including this previously characterized C/EBP-binding site was synthesized. In electrophoretic mobility shift assays (EMSAs), incubating this oligonucleotide together with nuclear extract from primary Clara cells isolated from rat, several shifts appeared (Figure 2A). These bands were efficiently 44
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FIG. 2. C/EBP-binding to the CYP2B1 promoter. (A) Electrophoretic mobility shift assay using nuclear extract from primary rat Clara cells incubated with an end-labeled oligonucleotide spanning the C/EBP-binding site in the CYP2B1 promoter (lane 1) and competed with a 200-fold excess of unlabeled homologous oligonucleotide (lane 2). In the presence of C/EBP␣ antibody, a super-shift appeared (lane 3) while inclusion of C/EBP␦ antibody did not alter DNA–protein interaction pattern (lane 4). (B) Western blot analysis for C/EBP␦ using Clara cell nuclear extract (lane 1) and nuclear extract from COS cells overexpressing C/EBP␦ (lane 2). Positions of molecular weight markers are indicated at left. (C) Labeled oligonucleotide was used for electrophoretic mobility shift assays with nuclear extracts from COS cells overexpressing C/EBP␣ (lane 4) or C/EBP␦ (lane 2) or C/EBP␦ together with anti-C/EBP␦ antibody (lane 3). As a control, nuclear extract from null-transfected COS cells was incubated with labeled oligonucleotide (lane 5). In lane 1, no nuclear extract was included. Indicated are specific retarded complex from rat Clara cells (I), super-shifted complex in the presence of antibody (ss), C/EBP␣ and ␦ containing complexes (␣ and ␦, respectively) and migration of free unbound oligonucleotide (free).
2B). To determine whether overexpressed C/EBP␦ was capable of interaction with the C/EBP-binding site in the CYP2B1 promoter, additional EMSAs were performed using COS cell nuclear extract. In the presence of C/EBP␣ or C/EBP␦ containing nuclear extracts, prominent DNA-protein interactions were demonstrated (Figure 2C). The C/EBP␦ shift could be supershifted upon inclusion of C/EBP␦ antibody.
results in a major decrease of promoter activity in transient transfection studies, the phenobarbitalinduction of the promoter is not affected by inactivation of the site (10 –12). Together these results suggest that C/EBP factors are major contributors to basal CYP2B1 expression and are not important for the phenobarbital induction of CYP2B1 in the liver. In the
Inactivation of the C/EBP-Binding Site in the CYP2B1 Promoter Results in Markedly Reduced Transactivation of C/EBP␣ and C/EBP␦ To determine the functional importance of the characterized C/EBP-binding site in lung epithelial cells, site-directed mutagenesis studies were performed. In transient transfection studies using a construct including the ⫺1350 bp proximal CYP2B1 promoter with the C/EBP-binding site inactivated, a marked reduction of the transactivation efficiency was seen from both C/EBP␣ and C/EBP␦ (Figure 3). This indicates that this C/EBP-binding site is the major site conferring transactivation of the CYP2B1-gene by C/EBP transcription factors in lung epithelial cells. FIG. 3. Functional analysis of the C/EBP-binding site in CYP2B1 promoter by cotransfection studies using a promoterreporter gene construct altered by site-directed mutagenesis. The ⫺1350 CYP2B1 promoter--galactosidase construct was subjected to site-directed mutagenesis in order to alter the DNA sequence of the C/EBP-binding site. pCMVC/EBP␣ or pCMVC/EBP␦ expression plasmids were cotransfected in A549 lung cells with constant amounts of promoter-reporter gene construct with wild-type (wt) or mutated (mut) promoter sequence. Transfection efficiencies were normalized as in Figure 1. Error bars represent SEM (n ⫽ 6).
DISCUSSION In this study we demonstrate that C/EBP␣ and C/EBP␦ regulate the CYP2B1 gene through interaction with a proximal C/EBP-binding site in lung epithelial cells. This C/EBP-binding site has previously been characterized in liver cells (11). Although it has been demonstrated that mutation of this C/EBP-binding site 45
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lung, CYP2B1 is constitutively expressed in bronchiolar Clara cells and at lower levels in alveolar type II cells. C/EBP␣ expression has been demonstrated in the alveolar region and at lower levels in the bronchiolar epithelium (18, 19). In contrast, C/EBP␦ is expressed at high levels in bronchiolar Clara cells and at lower levels in the alveolar region (18). In this study, we demonstrate that C/EBP␦ exhibits a higher transactivation potential compared to C/EBP␣ of the CYP2B1 gene. A higher transactivation potential for C/EBP␦ was evident from the transfection studies in both the A549 cells and in the Clara cell-like NCI-H441 cells. However, it is possible that the lower transactivation potential seen for C/EBP␣ in H441 cells reflects endogenous C/EBP␣ expression. In line with these results, it is possible that the higher levels of C/EBP␦ detected in the bronchiolar epithelium might explain the higher levels of CYP2B1 seen in this region. Moreover, in the liver, the levels of C/EBP␦ are low under normal conditions (20) perhaps explaining the low levels of CYP2B1 seen in this organ under non-induced conditions. CYP2B1 is a major enzyme responsible for the activation of tobacco-specific carcinogens in lung cells (21). The demonstration of a role for C/EBP factors in the regulation of pulmonary expression of CYP2B1 may thus help us understand mechanisms affecting susceptibility of the lung to injury from tobacco smoke. In EMSAs, no interaction of C/EBP␦ with the C/EBPbinding site in the CYP2B1 promoter was detected in rat primary Clara cell nuclear extract. To exclude the possibility that the C/EBP␦ isoform was incapable of interaction with the C/EBP-binding site in the CYP2B1 promoter, EMSAs were performed using nuclear extract from COS cells overexpressing C/EBP␦. These experiments demonstrated a strong interaction of C/EBP␦ with the CYP2B1 promoter. The finding that no interaction of C/EBP␦ was seen in the primary Clara cell nuclear extracts was unexpected as this protein has been shown to be highly expressed in murine lung and in Clara cells specifically (18, 20). Moreover, in Western blotting experiments it was shown that C/EBP␦ was present in the rat Clara cell nuclear extract. Together these data suggest that C/EBP␦ is prevented from interaction with the CYP2B1 promoter in nuclear extract from primary Clara cells. Previously it has been demonstrated that C/EBP␦ is regulated in its DNA-binding properties by phosphorylation in that it is posttranslationally modified by a member of the signal-transducing protein kinase cascade (22). Thus, it is possible that C/EBP␦ is modified in a way that prevents DNA-binding in the primary Clara cells. Furthermore, a change in phosphorylation of C/EBP␦ could explain the small size difference that was observed in the Western blot analysis between C/EBP␦ originating from primary Clara cells and overexpressed C/EBP␦ from COS cells. However, it can not be excluded that dephosphorylation of C/EBP␦ occur dur-
ing the Clara cell isolation process and thus these results do not necessary reflect the in vivo situation. C/EBP factors play important roles in controlling differentiation and differentiation-dependent processes. In many tissues, most notably in liver and fat, C/EBP factors are important regulators of proliferation, cell cycle arrest and gene expression (15). In rat lung, C/EBP␣ is expressed in a differentiationdependent pattern and is first detected around embryonic day 17–18 (19). Thus, the expression of C/EBP␣ parallels the cellular differentiation of the lung which takes place late during development resulting in highly specialized cell types such as the Clara cells of the conducting airways and the alveolar type II cells. The differentiation of these cell types is accompanied by expression of differentiation markers such as surfactant proteins, the Clara cell secretory protein (CCSP) and different P450 enzymes. In rat lung, CYP2B1 expression is detected late in development, around day 4 postnatal age (7, 8). We have previously shown that C/EBP factors are important for the regulation of the differentiation-dependent gene CCSP (18, 23). The results in the present study, indicating a role for C/EBP␣ and C/EBP␦ in the expression of CYP2B1, suggest that C/EBP factors have a more general role in differentiation-dependent gene expression in the lung epithelium. ACKNOWLEDGMENTS We are grateful to Per E. Schwarze, Marit Låg, and their group for isolation of primary rat Clara cells. We thank Rune Toftgård for the kind gift of reagents and Lena Nordlund-Mo¨ller for expert technical assistance. This work was supported by the Swedish Medical Research Council (Grant 13115), Swedish Match (Grant 199831), the Swedish Medical Society, the Swedish Heart–Lung Foundation, the research foundations “Tore Nilssons stiftelse fo¨r medicinsk forskning,” “Stiftelsen Lars Hiertas minne,” “Stiftelsen Sigurd och Elsa Goljes minne,” “Stiftelsen cystisk fibros forskningsfond,” and the Research Foundations of the Karolinska Institute.
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