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INTRODUCTION |
Signaling via the retinoic acid
(RA)1/retinoic acid receptor
(RAR) axis is known to be important to epithelial cell differentiation and proliferation in the lung (1). RAR signaling is required in lung
morphogenesis as observed in double knockout RAR
/
and RAR
/
mice that developed lung hypoplasia and aplasia (2). RA
influenced branching morphogenesis and alveolarization of the fetal
lung in vitro (3-5). RA enhanced SP-B mRNA and
surfactant protein B (SP-B) expression in lung epithelial cells and
explant cultures of fetal lungs (3, 5-8). SP-B is produced in alveolar type II epithelial cells and in subsets of non-ciliated bronchiolar cells lining conducting airways, and in H441 cells (human pulmonary adenocarcinoma cells). SP-B enhances the spreading and stability of phospholipids in surfactant in the alveoli and plays a critical role
in lamellar body and tubular myelin organization (9). SP-B is essential
for postnatal respiratory adaptation after birth. Mutations of the SP-B
gene in the human and mouse cause lung dysfunction at birth and
susceptibility to oxygen toxicity (10-13).
An enhancer located at -500/-375 base pairs was identified in
the hSP-B 5'-flanking regulatory region that contains clustered retinoic acid-responsive elements (RAREs) and TTF-1 binding sites (14,
15). Deletion of the enhancer sequence significantly reduced
transcriptional activity of the hSP-B promoter (14). These sites were
required for RA stimulation of hSP-B gene expression in respiratory
epithelial cells. Both RAR and TTF-1 bound to the clustered RARE and
TTF-1 binding sites in the enhancer region of the hSP-B gene (14, 15).
A dominant negative RAR mutant inhibited hSP-B transcription (16).
RAR belongs to the steroid/nonsteroid nuclear hormone receptor
superfamily and consists of three receptor isotypes
,
, and
,
which are encoded by distinct genes. RAR forms a heterodimer with
retinoid X receptor (RXR) that binds to RARE on the target genes. Whereas RAR has weak DNA binding affinity, RXR greatly enhances
RAR DNA binding affinity through dimerization of RAR/RXR (17). RAR
consists of a DNA binding domain containing Zn2+ finger
motifs, a ligand-binding/dimerization domain, a ligand-independent AF-1 transcription activation domain, and a
ligand-dependent AF-2 transcription activation domain.
Through these various functional domains, RAR interacts with other
transcription factors and coactivators to stimulate gene transcription.
TTF-1 is a tissue-specific transcription factor of Nkx2 family members
expressed in the lung, the thyroid, and part of the forebrain (18). In
the lung, TTF-1 mRNA and protein were detected at the earliest
stages of differentiation and were restricted to bronchial and alveolar
epithelium in the postnatal lung (18, 19). TTF-1 binds to and activates
the promoters of a number of genes selectively expressed in the
respiratory epithelium, including SP-B (20). Lung morphogenesis and
surfactant protein expression were markedly disrupted in TTF-1
/
mice (21). Studies by deletion/truncation mutagenesis, mammalian cell
cotransfection, electrophoretic mobility shift assay, and
immunofluorescent assays revealed three distinct functional domains of
TTF-1 (22). The N- and C-terminal regions of TTF-1 are transactivation
domains. The homeodomain (HD) of TTF-1 is responsible for DNA binding
and nuclear localization.
RA treatment triggers formation of an enhanceosome in the hSP-B
enhancer region that contains RAR/RXR, TTF-1, CBP, and p160 coactivators (19). The DNA binding of RAR and TTF-1 to the hSP-B enhancer plays a critical role for enhanceosome formation. Because both
clustered RAR and TTF-1 DNA binding sites overlap in the enhancer
region of the hSP-B gene, it is highly possible that RAR and TTF-1
interact with each other to facilitate enhanceosome formation. In this
report, direct interactions between TTF-1 and RAR
were identified
that enhanced DNA binding to the hSP-B enhancer in respiratory
epithelial cells.
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MATERIALS AND METHODS |
Cell Culture--
Human pulmonary adenocarcinoma cells (H441)
were cultured in RPMI supplemented with 10% fetal calf serum,
glutamine, and penicillin/streptomycin. Cells were maintained at
37 °C in 5% CO2/air and passaged weekly.
Co-localization of RAR and TTF-1 in H441 Cells by
Immunofluorescent Double Staining Assay--
The hRAR
(from Dr.
Pierre Chambon) and TTF-1-FLAG (22) expression vectors were
cotransfected into H441 cells. Immunofluorescent staining was performed
2 days after cotransfection following a procedure described previously
(22). TTF-1-FLAG was recognized by FLAG monoclonal antibody (Eastman
Kodak Co.) conjugated with fluorescein isothiocyanate, whereas hRAR
was recognized by hRAR
polyclonal antibody (Santa Cruz
Biotechnology, Santa Cruz, CA) conjugated with Texas red. The
fluorescent signals were analyzed by the Leica DM IRBE confocal microscope.
Transfection and Reporter Gene Assays--
The hSP-B-500
construct was made previously (14). The RAR
expression vector was
from the original authors (Dr. Pierre Chambon). For the RAR/TTF-1
cotransfection study, transient transfection and luciferase reporter
assays were performed as described previously (14, 15). Briefly, H441
cells were seeded at densities of 2 × 105 cells/well
in six-well plates. The hSP-B500 reporter construct (0.25 µg) was
cotransfected with 0.5 µg of RAR, 0.5 µg of PCR3.0/TTF-1, and 0.5 µg of pCMV-
-galactosidase plasmid into H441 cells by Fugene6
(Roche Molecular Biochemicals). After 2 days of incubation, cells were
lysed, and luciferase activities were performed using the luciferase
assay system (Promega). The light units were assayed by luminometry
(Monolight 3010, Analytical Luminescence Laboratory, San Diego, CA). In
each transfection,
-galactosidase activities were determined for
normalization of transfection efficiency.
Glutathione S-Transferase (GST) Pull-down Assay--
The
full-length and various truncated TTF-1 constructs were from a previous
study (22). The C-terminal domain was subcloned into the PCR3.0 vector
(Invitrogen, San Diego, CA) at the HindIII and
NotI sites. Because the TTF-1 C-terminal domain only
contains one methionine and generated a weak pull-down signal, three
extra methionine codons were placed before the NotI site by
PCR to enhance radioactive visualization (22).
To make GST fusion proteins, the full length, DBD, and AF-2 domain of
RAR
were subcloned into the pGEX4T-1 GST vector (Amersham Pharmacia
Biotech) at the EcoRI and NotI restriction enzyme
sites by PCR. The plasmids were transformed into JM109 or BL21
bacterial strains for protein expression. After 3 h induction at
37 °C by 1 mM
isopropyl-
-D-thiogalactopyranoside, the bacteria
were harvested and resuspended in 1× PBS, followed by sonication and
treatment with 1% Triton X-100. The proteins were purified by
incubation with a 50% slurry of glutathione-Sepharose 4B beads
(Amersham Pharmacia Biotech) for 30 min at room temperature and then
eluted from beads using glutathione elution buffer followed by
dialysis. Protein expression was confirmed by Coomassie Blue staining
and Western blots using either RAR
antibody or GST antibody
(Amersham Pharmacia Biotech) after gel electrophoresis. Protein
concentrations were determined.
For GST pull-down, full-length TTF-1 and various domain proteins
were synthesized and [35S]methionine (Met) labeled
using Promega's in vitro transcription/translation kit.
Approximately 1 µg each of purified GST, RAR
-GST,
RAR
DBD-GST, and RAR
AF-2-GST were incubated with 20 µl of 50%
glutathione-Sepharose 4B beads at room temperature for 30 min.
Approximately 25 µl of the [35S]Met-labeled full-length
TTF-1 and various domains were added to the fusion protein-bead mixture
and incubated at room temperature for 1.5 h. The
protein-protein-bead mixtures were washed three times with 30 µl of
1× PBS. The protein-protein-bead complexes were then resuspended in 30 µl of 1× SDS sample buffer and run on 10-20% tricine
polyacrylamide gels (Invitrogen-Novex). The protein gels were fixed and
incubated in Amplify reagent (Amersham Pharmacia Biotech) at
room temperature. The gels were dried and exposed to x-ray films for visualization.
Mammalian Two Hybrid System Assay--
The reporter pG5LUC,
pVP16/TTF-1 BD, and pM/RAR
BD constructs were made
previously (15). The plasmid constructs of pVP16/TTF-1 HD AD and
pVP16/RAR
DBD AD were made by subcloning the PCR products of TTF-1
HD and RAR
DBD into the pV16 AD vector
(CLONTECH, Palo Alto, CA) at the
EcoRI/XbaI sites. Transfection and luciferase assay were performed as described previously (15).
Electrophoretic Mobility Shift Assay--
Wild type
double-stranded Ba (-439 to -410) and Bb (-417 to -390) oligos from
the hSP-B enhancer region were synthesized, annealed, and purified as
described previously (14, 15). The oligonucleotides were radiolabeled
by [
-32P]ATP and polynucleotide kinase, and incubated
with 10 ng of the purified TTF-1 HD-GST fusion protein and 100 ng of
the RAR
DBD-GST fusion protein alone or in combination.
Electrophoretic mobility shift assay was performed by following the
procedures described previously (14, 15). As a negative control, GST
protein was also incubated with the radiolabeled probes alone or in
combination with TTF-1 HD-GST or RAR
DBD-GST.
Chromatin Immunoprecipitation Assay of RAR
and TTF-1 in H441
Cells--
The assay followed a previously published procedure (23).
Briefly, H441 cells were seeded at a density of 1 × 106 cells per 100-mm dish. The following day, cells
were treated with 10 µM of all trans retinoic
acid for 24 h. Untreated cells served as controls. Cells were then
treated with 1% formaldehyde in serum-free RPMI for 10 min at room
temperature to cross-link proteins and DNA, followed by rinsing with
PBS twice. The cell pellets were collected by centrifugation. The cells
were lysed by adding 150 µl of lysis buffer (25 mM Tris,
pH 8.1, 140 mM NaCl, 1% Triton X-100, 0.1% SDS, 3 mM EDTA, and 1× protease inhibitor mixture (Roche
Molecular Biochemicals) and were allowed to incubate on ice for 10 min.
The cells were sonicated followed by centrifugation, and the
supernatants containing soluble chromatin were collected. For
immunoprecipitation, either 20 µl of 100 µg/ml RAR
(Santa Cruz
Biotechnology), 5 µl of monoclonal TTF-1 antibody, or 1 µl of
monoclonal FLAG antibody (Kodak) were added to the supernatants and
incubated overnight at 4 °C. The sonicated salmon sperm DNA (5 µg)
was also added to cell lysate-antibody complex. Fifty µl of
50% protein A/G plus agarose beads (Santa Cruz Biotechnology) were
added to the samples and incubated at 4 °C for 2 h followed by
centrifugation. The pellets were washed three times, once by 100 µl
of TSE 150 mM NaCl buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 150 mM NaCl), once by100 µl of TSE 500 mM NaCl
buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, and 500 mM NaCl) and once
by 100 µl of Buffer III (0.25 M LiCl, 1% Nonidet P-40,
1% deoxycholate, 1 mM EDTA, and 10 mM
Tris-HCl, pH 8.1). Each wash was performed on ice for 10 min. Next, the
samples were washed three times with 100 µl of TE buffer. The
immunocomplexes were then eluted off the beads by incubation with 1%
SDS and 0.1 M NaHCO3 on ice for 10 min. Samples
were heated at 65 °C for 4 h to reverse formaldehyde cross-linking followed by phenol-chloroform extraction and
sodium acetate/ethanol precipitation. The DNA was then used as
templates for quantitative PCR analysis with primers corresponding to
the SP-B enhancer region.
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RESULTS |
Co-localization of RAR
and TTF-1 in H441 Cells by
Immunofluorescent Double Staining Assay--
To study whether RAR
and TTF-1 interact with each other to regulate hSP-B gene expression,
RAR
and TTF-1 colocalization was assessed in H441 cells by double
immunofluorescent staining. The hRAR
and TTF-1-FLAG expression
vectors were cotransfected into H441 cells. Expression of TTF-1-FLAG
protein (fluorescein isothiocyanate) and hRAR
protein (Texas red)
was monitored by immunofluorescent staining using FLAG and RAR
antibodies. Confocal microscope analysis of fluorescent image revealed
colocalization of RAR
and TTF-1-FLAG proteins in the nucleus of H441
cells (Fig. 1).

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Fig. 1.
Colocalization of RAR
and TTF-1 in the nucleus of H441 cells. A, a
nucleus of a H441 cell; B, fluorescein isothiocyanate
immunofluorescent staining of TTF-1-FLAG expression by monoclonal
antibody recognizing the FLAG sequence; C, Texas red
immunofluorescent staining of hRAR expression by polyclonal antibody
recognizing RAR; D, overlay of fluorescein isothiocyanate
and Texas red immunofluorescent staining by confocal microscope.
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Synergistic Stimulation of the hSP-B500 by RAR
and
TTF-1--
Overlapping of clustered RAR
and TTF-1 DNA binding sites
located in the 5'-flanking region of the hSP-B gene provides the possibility that two proteins may interact with each other to synergistically stimulate hSP-B promoter expression. RAR
and TTF-1
expression vectors were cotransfected with the hSP-B500 luciferase
reporter gene into H441 cells. The stimulatory effect of double
transfection of RAR
and TTF-1 was much higher than the additive
effect of RAR
and TTF-1 transfection alone (Fig. 2), indicating that RAR
and TTF-1 may
interact to stimulate the hSP-B promoter in respiratory epithelial
cells.

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Fig. 2.
Synergistic stimulation of
RAR and TTF-1 on the hSP-B 500. The
luciferase reporter construct hSP-B 500 (0.25 µg) was cotransfected
with 0.5 µg of TTF-1, RAR , and TTF-1/RAR constructs into H441
cells, respectively. Cells were harvested, and luciferase activity was
measured 72 h later. The activity of hSP-B 500 without
cotransfection was defined as 1. Values shown are means ± S.D.;
n = 3.
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Protein-Protein Interaction between RAR
and TTF-1 by GST
Pull-down Study--
To prove a direct interaction between RAR
and
TTF-1, a GST pull-down assay was performed. RAR
was fused with GST
to make a fusion protein. The purified RAR
-GST fusion protein was
incubated with the [35S]methionine labeled TTF-1 protein.
After purification of incubated protein complexes through a Sepharose
4B-glutathione column, the radiolabeled TTF-1 protein was
retained by the RAR
-GST fusion protein as detected by polyacrylamide
gel electrophoresis and autoradiography (Fig.
3). The GST control alone showed a very weak nonspecific pull-down signal, whereas the empty vector control (PCR3.0) showed no signal at all, indicating that
[35S]Met-TTF-1 pull-down by the RAR
-GST fusion protein
was specific. This indicates the direct protein-protein interaction
between RAR
and TTF-1 in vitro. The interaction between
RAR
and TTF-1 was not further enhanced by RA treatment (data not
shown), suggesting that the ligand-dependent AF-2 domain
may not be required for interaction with TTF-1.

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Fig. 3.
Protein-protein interaction between
RAR and TTF-1. Pull-down of full-length
[35S]Met-TTF-1 by RAR -GST fusion protein. GST was used
as a control. PCR3.0, an empty expression vector.
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TTF-1 HD Interaction with RAR
--
To better understand the
mechanism by which RAR
and TTF-1 interact with each other, specific
domains of TTF-1 required for RAR
interaction need to be defined.
Previously, TTF-1 functional domains were characterized (Fig.
4A) (22). The N- and
C-terminal domains of TTF-1 were transactivation domains for hSP-B
promoter activation. The HD of TTF-1 was the DNA binding and
nuclear localization domain. Different portions of the TTF-1 molecule
were constructed and radiolabeled with [35S]methionine.
The radiolabeled TTF-1 fragments were incubated with the full-length
RAR
-GST fusion protein for pull-down. TTF-1 N-terminal and
C-terminal domains failed to be pulled down by RAR
-GST, whereas the
TTF-1 HD was successfully pulled down by RAR
-GST (Fig.
4B). Other TTF-1 fragments containing the HD were also
pulled down by RAR
-GST. Therefore, the TTF-1 HD is responsible for
protein-protein interaction with RAR
. The interaction appears to be
enhanced by the N-terminal domain of TTF-1.

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Fig. 4.
Identification of TTF-1 functional
domains required for RAR interaction.
A, schematic domains of TTF-1 and RAR . N-term,
TTF-1 N-terminal transactivation domain; HD, TTF-1
homeodomain; C-term, TTF-1 C-terminal domain;
AF-1, RAR ligand-independent activation domain;
DBD, RAR DNA binding domain; LBD, RAR
ligand binding domain; AF-2, RAR
ligand-dependent activation domain; F, RAR F
domain. B, various [35S]Met-TTF-1 fragments
containing the HD were pulled down by RAR -GST fusion protein. GST
was used as a control.
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RAR
DBD Interaction with TTF-1 HD--
RAR
is composed of
several functional domains, including AF-2, DBD, LBD, and AF-1 domains
(1). Because RAR and TTF-1 DNA binding sites overlap in the enhancer
region of the hSP-B gene, it is highly likely that RAR
DBD is
involved in the protein-protein interaction with TTF-1. The AF-2 domain
is required for ligand-dependent pull-down of nuclear
receptor coactivators (25, 29-31). Therefore, both RAR
DBD and AF-2
domains were selected for further study with TTF-1. Different portions
of the TTF-1 molecule were radiolabeled with
[35S]methionine. The radiolabeled TTF-1 fragments were
incubated with the purified RAR
-DBD-GST or RAR
-AF2-GST fusion
proteins for pull-down. The full-length TTF-1 and the TTF-1 HD were
pulled down by RAR
-DBD GST, but not by RAR
-AF2-GST (Fig.
5). This is in agreement with the
observation that TTF-1 pull-down by RAR
was not
RA-dependent. Therefore, DNA binding domains for both proteins are involved in protein-protein interaction in
vitro.

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Fig. 5.
Identification of
RAR functional domains required for TTF-1
interaction. Full-length [35S]Met-TTF-1 and the
TTF-1 HD were pulled down by RAR -DBD GST (1 µg).
N-term, TTF-1 N-terminal transactivation domain;
C-term, TTF-1 C-terminal transactivation domain;
HD, TTF-1 homeodomain; DBD, RAR DNA binding
domain; AF-2, RAR ligand-dependent activation
domain.
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Protein-Protein Interaction between RAR
and TTF-1 in the
Mammalian Two Hybrid System--
To confirm that RAR
DBD and TTF-1
HD are required for the interaction with partners in cells, a mammalian
two hybrid system was used. The pair of TTF-1 HD AD/RAR
BD
constructs (Fig. 6A) and the
pair of RAR
DBD AD/TTF-1 BD constructs (Fig. 6B) were co-transfected into H441 cells with the luciferase reporter construct pG5LUC. The luciferase activities were markedly increased in
paired cotransfection (Fig. 6). These findings further support
the concept that DNA binding domains of both RAR
and TTF-1 are
required for protein-protein interaction both in vitro and
in cells.

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Fig. 6.
Interaction between RAR
and TTF-1 in the mammalian two hybrid system.
A, the luciferase reporter pG5LUC (0.5 µg) was
cotransfected with the pairs of TTF-1 HD AD (0.5 µg)/RAR BD (0.5 µg) into H441 cells to monitor protein-protein interactions.
Individual transfection controls are TTF-1 HD AD (0.5 µg)/pM BD (0.5 µg) and pVP16 AD (0.5 µg)/RAR BD
(0.5 µg). Luciferase activity was measured 48 h later. Values
shown are means ± S.D.; n = 3. B, the
assay was performed as outlined in A, except that the
luciferase reporter pG5LUC (0.5 µg) was cotransfected with the pairs
of TTF-1 BD (0.5 µg)/RAR DBD AD (0.5 µg) into H441 cells.
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RAR
DBD Effect on TTF-1 HD DNA Binding Affinity--
The
protein-protein interaction between DNA binding domains of RAR
and
TTF-1 prompted us to exam how this interaction alters their DNA binding
affinity in the enhancer region of the hSP-B gene. Because the HD is
the DNA binding domain and is sufficient for TTF-1 binding to the
enhancer region of the hSP-B gene (14), the TTF-1 HD-GST fusion protein
was used for the DNA binding assay. The purified TTF-1 HD-GST and
RAR
DBD-GST fusion proteins were incubated with the oligo Ba (-439
to -410) from the enhancer region of the hSP-B gene, which contains
overlapping RARE and TTF-1 DNA binding sites as reported previously
(15), individually or in combination. The DNA-protein interactions were
monitored by electrophoretic mobility shift assay. The RAR
DBD-GST
fusion protein alone had no detectable DNA binding activity to
oligo Ba. This is in agreement with previous observations that DNA
binding activity of RAR
required dimerization with RXR.
Interestingly, the RAR
DBD-GST fusion protein significantly enhanced
the DNA binding affinity of the TTF-1 HD-GST fusion protein to oligo Ba
(Fig. 7). As a negative control, no
enhanced TTF-1 HD-GST DNA binding activity to Ba oligo was observed by
the GST protein after incubation with TTF-1 HD-GST (data not
shown).

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Fig. 7.
Enhancement of TTF-1 HD DBD binding affinity
by RAR DBD. The radiolabeled
oligonucleotide Ba probe was incubated with the purified TTF-1 HD-GST
fusion protein or the RAR -DBD GST fusion protein or in combination.
Free probes and DNA-protein complexes were separated on 4%
nondenaturing polyacrylamide gels. Arrows indicate the
TTF-1-HD-GST DNA complex. The core sequence for TTF-1 binding is
underlined, and that for RARE is
italicized.
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RA Enhances RAR and TTF-1 DNA Binding to the hSP-B 5'-Flanking
Regulatory Region in H441 Cells--
In vivo chromatin
immunoprecipitation assay was used to test whether RA treatment
enhances RAR and TTF-1 binding affinity to the 5'-flanking regulatory
region of the hSP-B gene in cells. The primers corresponding to hSP-B
base pair -500 to +41 fragment, which contains both clustered RAR and
TTF-1 sites, were used for PCR of immunoprecipitated chromatin. The
monolayer of H441 cells was treated with all-trans RA
(10
5 M). The untreated cells were
used as a control. After protein-DNA cross-linking, soluble chromatin
of H441 cells was prepared and immunoprecipitated with the RAR
antibody. The amount of the hSP-B DNA sequence associated with RAR was
examined by analytic PCR using paired primers corresponding to the
hSP-B enhancer and the promoter region (base pairs -500 to
+41). RA treatment enhanced RAR binding affinity to the hSP-B
5'-flanking regulatory region (Fig.
8A). When the TTF-1 antibody
was used to immunoprecipitate soluble chromatin, RA treatment also
enhanced TTF-1 DNA binding affinity to the hSP-B 5'-flanking regulatory
region (Fig. 8B). As a negative control, when the FLAG
antibody was used to immunoprecipitate soluble chromatin, RA treatment
did not generate the hSP-B -500/+41 band (Fig. 8C). Plasmid
hSP-B 500 was also used as a template for PCR using the same set of
hSP-B primers (Fig. 8C). Taken together, RA treatment of
H441 cells enhanced RAR/TTF-1 complex formation in the hSP-B regulatory
region to stimulate hSP-B gene expression in vivo.

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Fig. 8.
Chromatin immunoprecipitation assay of RAR
and TTF-1 DNA binding to the hSP-B 500 enhancer in
vivo. H441 cells were harvested with or without RA
(10 5 M) treatment. Soluble
chromatin was immunoprecipitated with RAR antibody (A) or
TTF-1 antibody (B) and FLAG antibody (C).
Coprecipitated DNA was analyzed by PCR using a pair of primers
corresponding to the hSP-B promoter/enhancer region (base pairs
-500 to +41). In C, column C is a positive
control and represents PCR products using the hSP-B 500 plasmid as a
template (control). MW, molecular weight.
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|
 |
DISCUSSION |
Pulmonary surfactant is synthesized and secreted primarily by type
II epithelial cells in the alveoli of the lung. Deficiency or
disruption of pulmonary surfactant causes respiratory distress syndrome. Surfactant proteins facilitate the spreading and enhance the
stability of phospholipids in the alveoli and play an important role in
host defense. Transcriptional regulation of surfactant protein genes by
hormones and tissue-specific transcription factors is the key
step for elucidation of surfactant homeostasis in lung development and
postnatal respiratory adaptation.
RA was shown to be important for the stimulation of hSP-B gene
expression at the transcriptional level. Although the RA/RAR signaling
pathway is well known to be critical to epithelial cell differentiation
and proliferation in many tissues, little is known about how this
pathway interacts with and is determined by tissue-specific factors in
the respiratory system. We previously demonstrated that in the
pulmonary epithelial system, RA stimulation of the hSP-B promoter
through RARE sites is dependent on the juxtaposed clustered TTF-1 sites
in the enhancer region of the hSP-B gene (15). Both RAR
and TTF-1
were expressed in H441 cells and stimulated the hSP-B promoter in
dose-dependent fashions (7, 15). After separation from the
downstream TTF-1 sites, the clustered RARE sites in the enhancer region
still rendered the SV40 promoter response to stimulation by RA (15).
Therefore, the enhancer of the hSP-B gene works as an independent unit
in which the tissue-specific factor TTF-1 determines RA/RAR signaling
activity in pulmonary epithelial cells.
To determine whether RAR and TTF-1 directly interact with each other,
protein-protein interaction studies were performed in the present
study. There are three isotypes of RAR mediating RA function in cells.
Only RAR
was detected both in mouse type II epithelial cells and in
the H441 cell line that shares characteristics of non-ciliated
bronchiolar cells (7, 15). Therefore, RAR
was chosen for
protein-protein interaction with TTF-1 in H441 cells. GST pull-down
experiments demonstrated the direct protein-protein interaction between
the two proteins (Fig. 3). Deletion/truncation studies further defined
that the DBD domain of RAR
and the HD of TTF-1 were required for
protein-protein interaction (Figs. 4-6). Interestingly, the DBD domain
of RAR
enhanced TTF-1 HD DNA binding affinity on an hSP-B enhancer
oligonucleotide sharing the overlapping RARE/TTF-1 binding sites (Fig.
7). This may resulted from conformational changes of the
oligonucleotide or TTF-1 in the presence of RAR
DBD. This process
seems not be affected by RA. The ligand-dependent AF-2
domain, which is required for recruiting nuclear receptor coactivators
through protein-protein interaction, was not required for
protein-protein interaction with TTF-1. In addition, RAR
and TTF-1
were colocalized in the nucleus of H441 cells as demonstrated by
confocal/double immunofluorescent study (Fig. 1). Chromatin
immunoprecipitation study showed that RA treatment increased
recruitment of RAR and TTF-1 proteins to the enhancer region of the
endogenous hSP-B gene in H441 cells (Fig. 8). Collectively, our
data support the idea that RA/RAR not only depends on TTF-1 to
stimulate the hSP-B promoter but also facilitates TTF-1 binding to the
hSP-B enhancer region. TTF-1 and RAR together synergistically stimulate hSP-B transcription.
The complexity of RAR stimulation on target genes depends on multiple
protein factors interacting with its various functional domains. In
addition to interacting with TTF-1 in this study, RAR interacts with
many other transcription factors, including transcriptional
intermediary factor 2 (24, 25), AP-1 (26), TFIIH (27), and
TAFII135 (28), among others. Most importantly, RAR recruits
p160 nuclear receptor coactivators and CBP/p300 in the presence of RA
through physical interaction with the AF-2 domain (25, 29-31). We
previously demonstrated that CBP and p160 nuclear receptor coactivators
(steroid receptor coactivator 1, transcriptional intermediary factor 2, and activator of thyroid and retinoic acid receptor)
significantly stimulated the hSP-B promoter in a
dose-dependent fashion (15). p160 nuclear receptor coactivators and CBP interacted with TTF-1 in the mammalian two hybrid
system and synergistically stimulated hSP-B500 with TTF-1 (19). They
were all colocalized with SP-B in developing and adult epithelial cells
in the lung (19). Coiling of DNA around a histone octamer in the
nucleosome is a cornerstone of transcriptional control. Nucleosomes
repress all genes, including genes essential for respiratory functions.
They occlude sites of protein binding to DNA and interfere with the
interaction of activators, polymerases, transcription factors, and
DNA-modifying enzymes (32). Therefore, relief of repression by
chromatin is the first step toward the initiation of gene
transcription. p160 nuclear receptor coactivators and CBP/p300 possess
intrinsic histone acetyltransferase activity, which reversibly
acetylates specific lysine residues within the N-terminal tails of core
histones and leads to chromatin remodeling and gene activation
(32).
Based on our findings, an enhanceosome model was postulated that TTF-1,
RAR/RXR, CBP, and p160 coactivators form a transcriptional complex in
the enhancer region of the hSP-B gene (19), which may play an important
role in temporal and spatial expression of SP-B during lung
development. The interaction between RAR and TTF-1 provides a
foundation for the enhanceosome formation in the enhancer region of the
hSP-B gene. Because both RAR and TTF-1 are essential for lung
organogenesis and branching morphogenesis, our study provided direct
evidence and a model system to explain how RAR and TTF-1 interact with
and depend on each other to regulate lung-specific gene expression at
the transcriptional level to influence lung development.