Division of Pulmonary Biology, Children's Hospital Medical Center, Cincinnati, Ohio 45229-3039
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ABSTRACT |
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Retinoic acid (RA)
receptors (RARs) belong to the nuclear hormone receptor superfamily and
play important roles in lung differentiation, growth, and gene
regulation. Surfactant protein (SP) B is a small hydrophobic protein
synthesized and secreted by respiratory epithelial cells in the lung.
Expression of the SP-B
gene is modulated at the transcriptional and posttranscriptional
levels. In the present work, immunohistochemical staining revealed that
RAR- is present on day
14.5 of gestation in the fetal mouse lung. To assess
whether RAR is required for SP-B gene
transcription, a dominant negative mutant human (h) RAR-
403 was
generated. The hRAR-
403 mutant was transcribed and translated into
the truncated protein product by reticulocyte lysate in vitro. The
mutant retained DNA binding activity in the presence of retinoid X
receptor-
to an RA response element in the hSP-B promoter. When
transiently transfected into pulmonary adenocarcinoma epithelial cells
(H441 cells), the mutant hRAR-
403 was readily detected in the cell
nucleus. Cotransfection of the mutant hRAR-
403 repressed activity of
the hSP-B promoter and inhibited RA-induced surfactant
proprotein B production in H441 cells, supporting the
concept that RAR is required for hSP-B gene transcription in vitro.
human surfactant protein B; lung development; nuclear receptors
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INTRODUCTION |
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SURFACTANT PROTEIN (SP) B is a 79-amino acid amphipathic peptide produced by the proteolytic cleavage of surfactant proprotein (proSP) B in type II epithelial cells in the alveoli of the lung. The SP-B peptide is stored in lamellar bodies and secreted with phospholipids into the airway lumen. The function of SP-B is to stabilize the surfactant membrane layer and facilitate the spreading of phospholipids, preventing collapse during the respiratory cycle. SP-B is an essential component of surfactant and is required for postnatal respiratory adaptation (43). Mutations in the SP-B gene in both humans and mice cause respiratory failure after birth (8, 29, 30).
It has been well established that retinoic acid (RA) receptors (RARs)
play critical roles in proliferation, differentiation, and apoptosis in
a variety of epithelial cells. Recently, RAR- and -
and retinoid
X receptor (RXR)-
were detected in the H441 cell line, which is
derived from pulmonary adenocarcinoma epithelial cells. In H441 cells,
RA and RARs stimulated SP-B promoter activity, mRNA accumulation, and
protein production (12, 13, 45). Increases in SP-B mRNA and protein
accumulation in fetal lung explants have also been observed (2, 28). RA
stimulation of the human (h) SP-B gene
is mediated through direct binding of the RAR/RXR heterodimer to
the SP-B promoter (45). The region between
375 to
500 bp
of the hSP-B promoter was identified as the responsible sequence for
RAR binding and transactivation (45).
Lung development is dependent on expression of RARs (10, 26). RAR-,
-
, and -
belong to the superfamily of nuclear receptors. They
form heterodimers with the RXR, bind to the RA response element (RARE)
on promoter regions of target genes, and exert stimulatory effects
after binding to their ligand RAs (19, 21, 22). Expression of all three
isotypes of RAR was previously detected in the developing lung by in
situ hybridization or RT-PCR in the mouse and rat (10, 25). RAR-
and
-
null mutant mice died in utero and had severely hypoplastic lungs
(26). Lung organogenesis is dependent on interactions between
mesenchymal and epithelial cells. Several studies (3, 6, 28) in lung
buds indicated that lung branching morphogenesis and differentiation
were strongly influenced by RA in vitro. Recent studies (23, 24)
demonstrated that RA also influences alveolarization. Treatment of rats
with all trans-RA increased the number
of alveoli and reversed an alveolar disorder caused by elastase-induced
pulmonary emphysema in animals (23, 24).
RARs consist of a DNA binding domain that contains
Zn2+ finger motifs, ligand binding
and dimerization domains, a ligand-independent transcriptional
activation (AF-1) domain, a ligand-dependent transcriptional activation
(AF-2) domain, and an F region (19, 21, 22). Through these various
domains, RARs interact with other transcriptional and signaling
cofactors, including p160/SRC-I/TIF-II/Rac-III, CBP/p300, AP-1, TFIIH,
and TAFII135 (7, 15, 16, 18, 27, 31, 35, 39, 42). Through structure-function studies, dominant negative
mutants of RARs have been developed and characterized (9, 11). Removal
of the COOH-terminal AF-2 domain of RARs yields dominant negative
mutant receptors in cultured animal cells. The integrity of both the
DNA binding and heterodimerization functions of RARs is required for
the dominant negative effect. The dominant negative mutant of RAR-
blocks wild-type RAR function and has been successfully used in
transgenic mice (17).
To further elucidate the biological functions and mechanism of RAR-
in the regulation of hSP-B gene
expression, a dominant negative mutant, hRAR-
403, was generated and
expressed in H441 cells with a mammalian cell expression vector. This
was based on an observation that RAR-
was detected on
day 14.5 of gestation in the fetal
mouse lung. The mutant RAR-
protein was expressed in H441 cells and
localized to the nucleus. The mutant hRAR-
403 protein retained DNA
binding activity on the RARE of the hSP-B promoter. Cotransfection of
the mutant hRAR-
403 strongly inhibited transcription of the hSP-B
promoter by luciferase reporter assay and proSP-B production in H441 cells.
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METHODS AND MATERIALS |
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Cell culture. Human pulmonary adenocarcinoma (H441) cells were cultured in RPMI 1640 medium (GIBCO BRL, Grand Island, NY) supplemented with 10% fetal calf serum, glutamine, and penicillin-streptomycin. Cells were maintained at 37°C in 5% CO2-air and passaged weekly.
Plasmid constructs. The hSP-B
500-bp promoter (hSP-B-500) was made as previously described (46).
hRAR-403-FLAG was generated by PCR with synthetic oligonucleotide
primers, with an hRAR-
/pSG5 construct as a template (kindly
provided by Dr. Pierre Chambon, Institut de Génétique et de
Biologie Moléculaire et Cellulaire, Strassbourg,
France). The upstream primer with the
EcoR I site and the Kozak
sequence was 5'-GCGGAATTCGCCACCATGGCCAGCAACAGCAGCTCC-3'. The downstream primer with the Xba I
site and the FLAG sequence (underlined) was
5'-CTCGCTCTAGATTATCACTTGTCATCGTCGTCCTTGTAGTCCGGGATCTCCATCTTCAGCGT-3'. The PCR products were digested with
EcoR I and
Xba I restriction enzymes and ligated
with EcoR
I-Xba I-digested PCR3.0 luciferase reporter plasmid that contains the cytomegalovirus (CMV) promoter (Invitrogen, Carlsbad, CA). The correctness of hRAR-
403-FLAG/PCR3.0 was confirmed by DNA sequencing. hRAR-
462-FLAG/PCR was generated the
same way except that the downstream primer was
5'-CTCGCTCTAGATTATCA
CGGGGAGTGGGTGGCCGGGCT-3'. All primers were made by GIBCO BRL.
In vitro transcription, translation, and Western blot
analysis. hRAR-403-FLAG/PCR3.0 and
hRAR-
462-FLAG/PCR3.0 were transcribed and translated with the
TNT T7 Quick Coupled
Transcription/Translation System (Promega, Madison, WI) at 30°C for
90 min. As a positive control, thyroid transcription factor
(TTF)-1-FLAG/PCR3.0 was also transcribed and translated. The negative
control was the PCR3.0 empty vector. The programmed products were
separated on a 10% polyacrylamide gel and subsequently transferred to
a nitrocellulose membrane. Western blot analysis with an anti-FLAG
monoclonal antibody was performed as described previously (14).
Electrophoretic mobility shift assay.
A previously described RARE oligo probe of the hSP-B promoter was used
for an electrophoretic mobility shift assay (EMSA) study (45). In vitro
transcribed and translated hRAR-403 and hRXR-
proteins were
incubated with the radiolabeled probe and separated by 4%
nondenaturing gel as described previously (46). Antibody recognizing
RAR (5 µg; Santa Cruz Biotechnology, Santa Cruz, CA) was used for
identifying the hRAR-
403/hRXR-
complex in an EMSA study.
Transient transfection and luciferase
assays. Cotransfection of hRAR-403-FLAG with
hSP-B-500 and a luciferase assay were performed in H441 cells as
previously described (45, 47). The pCMV-
-Gal plasmid was
cotransfected for normalizing transfection efficiency. Each experiment
was repeated at least three times. Significance of the inhibitory
effects of hRAR-
403-FLAG was determined by one-way ANOVA with the
SigmaStat program.
Immunohistochemistry and immunofluorescent
staining. Immunohistochemical staining of H441 cells
transfected with hRAR-403-FLAG/PCR3.0, hRAR-
462-FLAG/PCR3.0, and
TTF-1-FLAG/PCR3.0 with the anti-FLAG antibody (Kodak, New Haven, CT)
was performed as previously described (45). In the control cells, no
specific antibody was added. Immunofluorescent staining of H441 cells
transfected without and with hRAR-
403-FLAG/PCR3.0 was performed as
previously described (14). Expression of hRAR-
403-FLAG/PCR3.0 was
detected with the anti-FLAG antibody and Texas Red-conjugated goat
anti-rabbit secondary IgG (Jackson ImmunoResearch Laboratories, West
Grove, PA). Expression of proSP-B was detected with a proSP-B
polyclonal antibody and FITC-conjugated goat anti-mouse IgG secondary
IgG (Jackson ImmunoResearch Laboratories). All
trans-RA was purchased from Sigma (St.
Louis, MO).
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RESULTS |
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Expression of RAR- in developing
lung. We previously detected RAR-
expression in
respiratory epithelial H441 cells. To assess which isoforms of RAR are
expressed in the developing lung, tissue sections from a fetal mouse
lung on day 14.5 of gestation were immunohistochemically stained with an antibody recognizing RAR-
. Figure 1 shows staining of RAR-
in the
epithelium of the developing mouse fetal lung by the anti-RAR-
polyclonal antibody. Based on this result, the
isotype of RAR was
chosen to make a dominant negative mutant for further study of the
hSP-B promoter.
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Construction of dominant negative
hRAR-403-FLAG. To test whether
expression of a dominant negative mutant of RAR-
inhibited the hSP-B
promoter, the mutant hRAR-
403-FLAG and the wild-type hRARa462-FLAG
were inserted into the mammalian expression vector PCR3.0 as
illustrated in Fig. 2. The mutant contains
the AF-1, DNA binding domain, and ligand binding and dimerization
domains of hRAR-
but lacks the AF-2 and F domains. A Kozak sequence
(ACCATGTCG) was included at the
NH2 terminus of hRAR-
403 and
hRAR-
462 to enhance the efficiency of translation. A FLAG sequence
was included at the COOH terminus of hRAR-
403 and hRAR-
462 and
was used to distinguish the mutant receptor from endogenous RAR-
.
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Expression of hRAR-403-FLAG and
hRAR-
462-FLAG in vitro. To test whether
the hRAR-
403-FLAG/PCR3.0 and the wild-type hRAR-
462-FLAG/PCR3.0 vectors generated protein products of correct sizes, the constructs were transcribed and translated in vitro in the rabbit reticulocyte lysate system. The products of the programmed lysates were separated on
a 10% polyacrylamide gel and analyzed by Western blot with the
anti-FLAG antibody. Both the full-length hRAR-
462-FLAG and the
short-form hRAR-
403-FLAG were produced in the
reticulocyte lysate system (Fig. 3).
Because TTF-1-FLAG/PCR3.0 was well characterized previously (14), it
was also transcribed and translated in vitro as a positive control. In
the negative control, the reticulocyte lysate revealed no FLAG fusion
protein.
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Expression of hRAR-403-FLAG in H441
cells. Expression of the hRAR-
403-FLAG and
hRAR-
462-FLAG vectors in H441 cells was assessed. The constructs
hRAR-
403-FLAG/PCR3.0, hRAR-
462-FLAG/PCR3.0, and TTF-1-FLAG/PCR3.0
(positive control) were transiently transfected into H441 cells.
Immunohistochemical analysis with an anti-FLAG antibody revealed strong
nuclear staining of the FLAG epitope for all three constructs (Fig.
4). Deletion of the AF-2 and F domains did
not change hRAR-
403 expression and nuclear localization in H441
cells. Untransfected H441 cells did not stain with the anti-FLAG
antibody.
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DNA binding activity of hRAR-403-FLAG
on RARE of the hSP-B promoter. Next, in vitro
transcribed and translated hRAR-
462-FLAG and hRAR-
403-FLAG fusion
proteins were assessed for their DNA binding activities. As shown in
Fig. 5, hRAR-
462-FLAG, hRAR-
403-FLAG, or hRXR-
-FLAG fusion protein alone did not form DNA-protein
complexes with RARE of the hSP-B promoter. This is due to their low DNA binding affinity. Yan et al. (45) previously reported that
when higher concentrations of purified bacteria-expressed
hRAR-
-glutathione S-transferase
(GST) fusion protein were used, interaction between hRAR-
-GST and
RARE was observed. In contrast, formation of
hRAR-
462-FLAG/hRXR-
-FLAG and hRAR-
403-FLAG/hRXR-
-FLAG
heterodimers with high DNA binding affinity generated detectable
DNA-protein complexes with RARE (Fig. 5). Binding of
hRAR-
462-FLAG/hRXR-
-FLAG and hRAR-
403-FLAG/hRXR-
-FLAG complexes with RARE was blocked by the anti-RAR antibody. This is
probably due to interference of the RAR antibody with either DNA
binding or dimerization of the hRAR-
403-FLAG/hRXR-
or
hRAR-
462-FLAG/hRXR-
heterodimer. Thus, like the wild-type
hRAR-
in the presence of hRXR, the mutant hRAR-
403 retained DNA
binding activity on RARE of the hSP-B promoter. Nonspecific bands
observed in EMSA, which did not contain the hRAR-
462-FLAG and
hRAR-
403-FLAG fusion proteins, were also detected in the control
samples (PCR3.0 empty vector). Nonspecific bands were not changed by
the RAR antibody.
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Inhibitory effect of hRAR-403-FLAG on
the hSP-B promoter in H441 cells. Cotransfection of
hRAR-
403-FLAG/PCR3.0 with the hSP-B-500 luciferase reporter gene
into H441 cells demonstrated that the mutant hRAR-
strongly
inhibited luciferase reporter activity (Fig.
6). In contrast, the wild-type hRAR-
stimulated the hSP-B luciferase reporter gene (45). The inhibitory
effects of the mutant hRAR-
403 on all
trans-RA-treated and untreated hSP-B-500 were dose dependent. Interestingly, the mutant hRAR-
did
not completely abolish the hSP-B-500 activity. The residual activity of
the hSP-B-500 seen in the presence of the mutant hRAR-
was similar
to that of the basal activity of the hSP-B 218-bp promoter luciferase
reporter gene, suggesting that RAR is not required for basal
transcription activity of the hSP-B promoter. Therefore, RAR stimulates
the hSP-B promoter through RARE located in the upstream enhancer region
as previously identified (46). All
trans-RA significantly reversed some
of the inhibitory effect of hRAR-
403. This was probably due to the
activation of endogenous RAR. Treatment with
9-cis-RA had the same reversal effect
as all trans-RA (data not shown).
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Inhibitory effect of hRAR-403-FLAG on
RA-induced proSP-B production in H441 cells. The effect
of hRAR-
403-FLAG on proSP-B was also examined in H441 cells with
double immunofluorescence staining analysis. All
trans-RA strongly stimulated proSP-B
synthesis in H441 cells as detected by a proSP-B polyclonal antibody
(Fig. 7A), in
agreement with previous observations that
9-cis-RA stimulated proSP-B staining
in H441 cells (45). This is also in agreement with the observation that
both all trans-RA and
9-cis-RA significantly increased SP-B
protein levels in human fetal lung explants (13). When cells were
transfected with hRAR-
403-FLAG as detected by a FLAG monoclonal
antibody, no stimulation of proSP-B by all trans-RA was
observed in transfected cells (Fig. 7,
B and
C).
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DISCUSSION |
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SP-B mRNA is expressed selectively in bronchiolar and alveolar cells,
and its expression is influenced at both the transcriptional and
posttranscriptional levels (1, 2, 4, 20, 32-34, 40, 41, 44, 46,
48). RA stimulates production of SP-B mRNA and increases
transcriptional activity (3, 12, 13, 28, 45). Stimulatory effects of RA
are mediated by direct DNA binding of liganded RAR/RXR to an
RARE on the hSP-B promoter (45). One approach to elucidate the
biochemical and physiological functions of RAR in the regulation of
SP-B gene transcription and
homeostasis is to utilize dominant negative RAR derivatives. To further
support the notion that RAR is required for full activation of the SP-B
promoter, a dominant negative hRAR-403-FLAG mutant was generated by
deleting the RAR-
AF-2 domain (Fig. 2). The RAR-
dominant
negative mutant was selected in the present study because
immunohistochemical staining indicated expression of RAR-
in the
fetal lung in vivo (Fig. 1) and in respiratory epithelial H441 cells in
vitro (45). It is worthwhile to notice that only a certain population
of progenitor epithelial cells was stained with the RAR-
antibody.
This may imply that this group of progenitor cells will have a
different fate during lung differentiation from cells without RAR-
expression.
The AF-2 domain of RAR is a ligand-dependent transactivation domain
located in the COOH-terminal part of RAR. The AF-2 domain is highly
conserved in many members of the nuclear receptor family and is
indispensable for the ligand-mediated function. This conserved domain
can be swapped between nuclear receptors without affecting the ligand
dependency for transactivation (19, 21, 22). The COOH terminus of the
domain (AF-2-AD) forms an amphipathic -helical structure (5). It is
proposed that hydrophobic residues of the AF-2-AD helix participate in
ligand binding and that the charged residues of the AF-2-AD helix
mediate protein-protein interactions with cofactors (see Ref. 16 for a
review). Point mutagenesis of the hydrophobic or charged residues of
this domain reduced its transactivation activity (11). After binding to RARE on the target genes, RARs interact with many coactivators (7, 15,
16, 18, 27, 31, 35, 39, 42). RARs and coactivators synergistically
interact with each other to stimulate gene transcription. The AF-2
domain of RAR plays essential roles in mediating protein-protein
interaction with coactivators. The present studies demonstrate that
deletion of the AF-2 domain abrogated the hRAR-
stimulatory effect
on the hSP-B promoter, suggesting that the AF-2 domain plays a critical
role in activation of the hSP-B promoter in respiratory epithelial cells.
RAR-, -
, and -
can be converted into potent dominant negative
transcriptional regulators that block the wild-type RAR function by
deletion of the AF-2 domain and actively repress the basal transcription level of target promoters (9). Although deletion of AF-2
domain abrogated RAR transactivation activity of the hSP-B promoter
(Fig. 6), it did not alter DNA binding of RAR to RARE of the hSP-B
promoter (Fig. 5). hRAR-
403 forms an inactive hRAR-
403/hRXR heterodimer that blocks formation of an active hRAR-
/hRXR
heterodimer to directly inhibit hSP-B
gene expression. The inhibitory effect of hRAR-
403 on the hSP-B
promoter might also result from indirect effects. This is evidenced by
the observation that all trans-RA significantly reversed the inhibitory effect of hRAR-
403 on the hSP-B promoter. The mutant RAR-
protein was readily detected after
transient transfection into H441 cells by immunohistochemical staining
(Fig. 4), suggesting its stability and appropriate translocation to the nucleus.
The finding that the SP-B gene is a downstream target of RAR in pulmonary respiratory epithelial cells is important for understanding the metabolism and homeostasis of SP-B in prenatal and postnatal development of the lung. The function of the newborn lung is dependent on the differentiation of respiratory epithelial cells and the synthesis and secretion of surfactant lipids and proteins into the air space. Pulmonary surfactant is composed of lipids and proteins that reduce surface tension at the air-liquid interface in the alveoli. SPs, including SP-A, SP-B, SP-C, and SP-D, are synthesized primarily by type II or bronchiolar epithelial cells and play critical roles in maintaining stability of the surfactant layer. Lack of pulmonary surfactant leads to alveolar collapse and epithelial cell lysis in respiratory distress syndrome, a major cause of morbidity and mortality in preterm infants. Bronchopulmonary dysplasia (BPD) is a chronic lung disease that often occurs in preterm infants as a result of prolonged and high inspired oxygen concentrations, barotrauma from mechanical ventilation, hyaline membrane disease, and secondary infection with prolonged tracheal intubation. Studies (36-38) have shown that vitamin A supplementation from the early postnatal period could reduce the morbidity associated with BPD in preterm infants. Studies (45; present study) showed that the vitamin A derivatives RA and RAR stimulate SP-B gene and protein expression in pulmonary epithelial respiratory cells, supporting the recent findings (23, 24) that RA plays a critical role in postnatal alveolarization in vivo.
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ACKNOWLEDGEMENTS |
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We thank Angela Naltner for critical reading of the manuscript. We
thank Xin Zeng for providing fetal lung tissue sections and Dr.
Jacquelyn A. Huffman Reed for helping with statistical analysis. We
thank Dr. Pierre Chambon for providing the human retinoic acid
receptor- and human retinoid X receptor-
plasmids.
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FOOTNOTES |
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This work was supported by the American Lung Association (C. Yan) and National Heart, Lung, and Blood Institute Specialized Center of Research Grant HL-56387 (to J. A. Whitsett and C. Yan).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: C. Yan, Children's Hospital Medical Center, Division of Pulmonary Biology, TCHRF, 3333 Burnet Ave., Cincinnati, OH 45229-3039.
Received 18 May 1998; accepted in final form 11 November 1998.
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