Department of Molecular Biology, University of Texas Health Science Center at Tyler, Tyler, Texas 75708-3154
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ABSTRACT |
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Surfactant protein B (SP-B) is essential for the
maintenance of biophysical properties and physiological function of
pulmonary surfactant. SP-B mRNA is expressed in a cell type-restricted
manner in alveolar type II and bronchiolar (Clara) epithelial cells of the lung and is developmentally induced. In NCI-H441 cells, a lung cell
line with characteristics of Clara cells, a minimal promoter region
comprising 236 to +39 nucleotides supports high-level expression of chloramphenicol acetyltransferase reporter
activity. In the present investigation, we characterized the upstream
promoter region,
236 to
140 nucleotides, that is
essential for promoter activity. Deletion mapping identified two
segments,
236 to
170 and
170 to
140
nucleotides, that are important for promoter activity. Mutational
analysis and gel mobility shift experiments identified thyroid
transcription factor-1, Sp1, and Sp3 as important trans-acting
factors that bind to sequences in the upstream promoter region. Our
data suggest that SP-B promoter activity is dependent on
interactions between factors bound to upstream and downstream regions
of the promoter.
lung; gene regulation; respiratory distress syndrome; transcription factors; surfactant protein B; thyroid transcription factor-1
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INTRODUCTION |
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SURFACTANT, A LIPOPROTEIN COMPLEX synthesized and secreted by alveolar type II epithelial cells, maintains the integrity of the alveoli during respiration by reducing surface tension at the alveolar air-tissue interface (14). Surfactant also plays important roles in host defense against certain bacteria and viruses through its actions to enhance phagocytosis by alveolar macrophages (32). Deficiency of surfactant is associated with newborn respiratory distress syndrome (2), the leading cause of neonatal morbidity and mortality in developed countries. Reduced levels and/or activity of surfactant may contribute to respiratory failure in adult respiratory distress syndrome (16). Surfactant protein (SP) B, a 9-kDa hydrophobic protein, is essential for the maintenance of biophysical properties and physiological function of surfactant. A number of studies have shown that SP-B promotes the adsorption and spreading of phospholipids (25) and stabilizes the phospholipid monolayer formed on the alveolar surface (10). Deficiency of SP-B due to a frame shift mutation in the coding region is associated with fatal respiratory failure in infants with congenital alveolar proteinosis (22). Targeted disruption of SP-B gene causes respiratory failure in newborn mice, further supporting the important role of SP-B in lung function (8).
Margana and Boggaram (19) previously found that a promoter
region containing the 236 to +39 nucleotides of the rabbit
SP-B gene is necessary and sufficient for high-level expression
of chloramphenicol acetyltransferase (CAT) reporter gene in
NCI-H441 cells, a human pulmonary adenocarcinoma cell line with
characteristics of bronchiolar epithelial cells (Clara cells). Further
deletion of the 5' region to the
140 nucleotide resulted
in ~80% reduction in promoter activity (19), suggesting that
the
236 to
140 region is required for full
expression of SP-B promoter activity. The minimal
promoter,
236 to +39 nucleotides, supported high-level CAT expression in a lung cell type-specific manner in
cell cultures, suggesting that it contained a cell- and/or
tissue-specific enhancer (19). A study by Margana and Boggaram (20)
also identified binding sites for Sp1, Sp3, thyroid transcription
factor (T TF)-1, and hepatocyte nuclear factor (HNF)-3 transcription
factors within the
140 nucleotide of the minimal SP-B
promoter that appeared to function in a cooperative or combinatorial
manner to maintain SP-B promoter activity. These data suggested
that the upstream promoter region,
236 to
140
nucleotides, contains functionally important cis-acting DNA
elements for which the identity remains to be determined.
The objective of our investigation was to identify and characterize
functionally important cis-acting DNA elements in the upstream
region (236 to
140 nucleotides) of the SP-B
minimal promoter. Deletion mapping of the upstream promoter region
identified two distinct regions,
236 to
170 and
170 to
140 nucleotides, that are important for
SP-B promoter activity. Mutational analysis and gel mobility
shift analysis of NCI-H441 nuclear proteins interacting with the
putative transcription factor binding sites identified functionally
important binding sites for TTF-1, Sp1, and Sp3 factors in the
SP-B upstream promoter region.
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MATERIALS AND METHODS |
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Nuclear extract preparation. Nuclear extracts from cells were
prepared according to the method described by Schreiber et al. (28).
Typically, cells from a confluent 75-cm2 flask were used
for preparation of the extract. The buffer composition of the final
nuclear extract was 20 mM HEPES, pH 7.9, 25% glycerol, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothrietol, 1 mM phenylmethylsulfonyl fluoride, 2.0 µg/ml each of leupeptin and aprotinin, and 0.5 mg/ml of
benzamidine. Nuclear extracts were divided into aliquots, which were
placed into chilled tubes, rapidly frozen in liquid nitrogen, and
stored at 80°C. The protein concentration of nuclear extract was determined by the method of Bradford (5) with Bio-Rad protein assay reagent.
Plasmid constructions and site-directed mutagenesis. SP-B
fragments 218 to +39 and
170 to +39 were amplified by PCR
with oligonucleotide sense primers
5'-CCCAGG
GA- GTCGG-3' and
5'-ACCAGC
AGGGGAG-3'
that correspond to
225 to
206 and
176 to
157
nucleotide sequences within SP-B promoter region and
an antisense primer,
5'-GCAGCCACG
GTGTGACTTGGCCGTGACG-3', that corresponds to +14 to +48 sequence of SP-B coding
region. The primers contained mutations (in boldface) that generated
Pst I and EcoR I sites (underlined) for cloning
purposes. The amplified fragments were ligated upstream of CAT
gene in the vector pSKCAT
S (1). pSKCAT
S containing the
SP-B promoter fragment
236 to +39 nucleotides served as
the template for site-directed mutagenesis by PCR according to the
method of Nelson and Long (21) as described (20). Oligonucleotides that
served as primers (sense) to introduce mutations into the transcription
factor binding sites in the SP-B promoter are shown in Table
1. The mutated DNA fragments
were inserted upstream of the CAT gene in pSKCAT
S. The
presence of introduced mutations in the insert DNA was established by
DNA sequencing.
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Electrophoretic mobility shift assays. Synthetic
oligonucleotides were annealed by heating 10 µM sense and antisense
oligonucleotides in 10 mM Tris · HCl 7.5, 10 mM
MgCl2, and 50 mM NaCl at 95°C for 5 min and then
allowing them to cool to room temperature over a period of 1 h. The
concentration of the double-stranded oligonucleotides was determined by
measuring absorbance at 260 nm (50 µg/ml = 1.0 A260
unit). Double-stranded oligonucleotides were end labeled with [-32P]ATP and T4 polynucleotide kinase.
Electrophoretic mobility shift assays (EMSAs) were performed by
incubating 0.5-1.0 ng (3-10 × 104
counts/min) of the oligonucleotide probe (Table
2) with 5 µg of nuclear protein in 20 µl of binding buffer [13 mM HEPES, pH 7.9, 13% glycerol, 80 mM
KCl, 5 mM MgCl2, 1 mM dithiothrietol, 1 mM EDTA, and 0.5 or
1 µg of poly(dI-dC) as nonspecific competitor] for 20 min at
30°C. Nuclear proteins were preincubated for 20 min at 30°C in
binding buffer before addition of the labeled probe. Competition
experiments were performed by addition of indicated molar excess of
cold wild-type or mutant oligonucleotides along with addition of the
labeled probe. In antibody supershift assays, nuclear extracts were
preincubated with 1.0 µg of preimmune IgG or polyclonal antibodies to
transcription factors for 1 h or overnight at 4°C before incubation
with the oligonucleotide probe. Four microliters of 6× loading
buffer [0.25% bromphenol blue, 0.25% xylene cyanol, and 15%
Ficoll (type 400) in H2O] were added to the
incubation mixtures before they were loaded onto a 6% nondenaturing polyacrylamide gel containing 0.5× Tris-borate-EDTA.
Electrophoresis was performed at constant current (30-35 mA) for
1.5-2 h with 0.5× Tris-borate-EDTA as running buffer. Gels
were vacuum-dried and exposed to autoradiographic film. Polyclonal
antibodies to human Sp1, Sp2, Sp3, and Sp4 and consensus and mutant Sp1
oligonucleotides were purchased from Santa Cruz Biotechnology.
Polyclonal antisera to the NH2-terminal portion of rat
thyroid-specific enhancer-binding protein (TTF-1) was kindly supplied
by Dr. Shioko Kimura (National Cancer Institute, Bethesda,
MD) and polyclonal antibodies to nuclear factor (NF)-1
(
-CTF1) were kindly supplied by Dr. Naoko Tanese (New York
University Medical Center, New York, NY).
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Cell culture and transfections. NCI-H441, a human pulmonary adenocarcinoma cell line with characteristics of Clara cells that expresses SP-B endogenously (23), was maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin B at 37°C in a humidified atmosphere of 5% CO2 and air. Cells were purchased from American Type Culture Collection (Manassas, VA).
Plasmids were amplified in Escherichia coli Top10F'
strain and purified by anion-exchange chromatography (QIAGEN, Valencia, CA), according to the manufacturer's protocol. The quality of the
plasmid DNA was analyzed by agarose gel electrophoresis and ethidium
bromide staining. Plasmid DNA preparations routinely contained
supercoiled DNA as the major DNA species. DNA concentration was
determined by measuring absorbance at 260 nm. At least two independent
preparations of plasmids were used for transfection. pCDNA3.1 (Invitrogen, Carlsbad, CA), a -galactosidase
expression vector, served as an internal control for normalization of
transfection efficiency. Plasmid DNAs were transiently transfected into
cells by liposome-mediated DNA transfer with LipofectAMINE (GIBCO BRL, Life Technologies, Gaithersburg, MD) as described previously (19). SP-B-CAT DNA (4.0 µg) and pCDNA3.1 (0.5 µg) were used to transfect cells plated on a 60-mm dish. Generally, CAT activity was determined after 48 h of incubation after DNA transfection.
CAT and -galactosidase assays. CAT activity of
cell extracts was determined by the liquid scintillation counting assay
(30) with [14C]chloramphenicol and
n-butyryl coenzyme A as described previously (19).
-Galactosidase activity was determined by chemiluminescent assay
with Galacto-Light Plus (TROPIX, Bedford, MA) substrate according to
the recommended protocol. Cell extracts were heat treated at 60°C
for 10 min or 48°C for 50 min to inactivate endogenous acetylase
and
-galactosidase activities before incubation with substrates. CAT
activities of cell extracts were normalized to cotransfected
-galactosidase activity to correct for variations in transfection efficiency.
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RESULTS |
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Deletion analysis of SP-B minimal promoter. A previous study by
Margana and Boggaram (19) showed that deletion of the 5' region of
SP-B minimal promoter from 236 to
140 nucleotides resulted in >80% loss of promoter activity. This suggested that the
promoter region extending from
236 to
140 nucleotides is required for full expression of SP-B promoter activity and may contain functionally important cis-DNA elements. To further map sequences in the upstream promoter region that are important for promoter activity, we generated SP-B-CAT promoter constructs
that contained deletions at the 5'-end and determined promoter
activity after transient transfection into NCI-H441 cells. Results
(Fig. 1) showed that deletion
of the 5' region of the minimal promoter to
170 and
140 nucleotides caused significant reduction in SP-B promoter activity, indicating that the upstream promoter regions,
236 to
170 and
170 to
140 nucleotides,
contained important cis-DNA elements. To further assess the
importance of these regions in SP-B promoter activity, we
analyzed the enhancer activities of segments of SP-B minimal
promoter (Fig. 2). SP-B fragments lacking the TATA sequence,
236 to
27,
236 to
140, and
140 to
27 nucleotides, were generated by
PCR and ligated upstream of the SV40 promoter in the vector
pSKSVCAT (1), and CAT expression was determined after transient
transfection into NCI-H441 cells. Results showed that SP-B
promoter fragments
236 to
140 and
140 to
27
increased CAT expression twofold compared with CAT expression by
SV40 promoter alone (Fig. 2). The enhancing activity of the
236 to
27 nucleotide region (~4-fold) was equivalent to
the combined activities of the
236 to
140 and
140
to
27 nucleotide regions (Fig. 2). These data further suggested
that the
236 to
140 and
140 to
27
nucleotide regions of SP-B minimal promoter contributed toward
enhancer activity and that contributions of both regions are required
for expression of SP-B promoter function. Consistent with the
results of the deletion mapping studies, the
236 to
140
nucleotide region displayed significant enhancing activity.
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Mutational analysis of SP-B upstream promoter region. Deletion
mapping experiments and analysis of enhancing activity showed that the
SP-B upstream promoter region, 236 to
140
nucleotides, is essential for promoter function. We searched the
upstream promoter region for the presence of putative transcription
factor binding sites using a transcription factor database (MacDNASIS,
Hitachi Software, San Francisco, CA) and by comparison with the
consensus binding sequences for the vertebrate-encoded transcription
factors (12). SP-B upstream promoter region contained putative
binding sites for Ets, activator protein-2, Sp1, NF-1 and TTF-1
transcription factors (Table
3). We investigated the
functional importance of the transcription factor binding sites by
mutational analysis (Table 1). Nucleotides in the binding sites were
altered by substitution mutagenesis, and the effect of mutations on CAT
expression was determined after transient transfection. Sp1 sites
were mutated by GG to TT substitution in the 5' portion
of the binding site. Mutation of these nucleotides has been shown to
drastically reduce the binding of Sp1 to its binding site (15). TTF-1
binding site was mutated by substituting nucleotides in the 3'
portion of the binding sequence. A previous study by Margana and
Boggaram (20) showed that alteration of nucleotides in the 3'
portion of the TTF-1 binding sequences at
112 and
102
nucleotides of SP-B promoter significantly reduced binding of
TTF-1, leading to reduction of promoter activity. Ets binding site was
mutated by GAA to CAG substitution. Similar mutations caused a
significant reduction in the promoter activity and binding of Ets
immunoreactive protein to Ets response element in the human
HER2 gene (29). In all cases, mutations of binding sites caused
significant reduction in binding of transcription factors as evaluated
by EMSA. Results (Fig. 3) showed that
mutations of Ets (
229 and
192), activator protein-2
(
225), NF-1 (
181), and Sp1 (
155) nucleotide sites did not have any significant effect on SP-B promoter activity, whereas
mutations of TTF-1 (
187), Sp1 (
162), or Ets (
149)
nucleotides caused an ~50% decrease in promoter activity.
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Identification of transcription factors binding to cis-DNA elements
in the upstream promoter region. Mutational analysis of putative
transcription factor binding sites in the upstream promoter region
identified that TTF-1 (187), Sp1 (
162), and Ets
(
149) binding sites serve important roles in SP-B
promoter function. We performed EMSA using NCI-H441 nuclear extracts to
identify proteins binding to the cis-DNA elements. EMSA with
SP-B promoter oligonucleotide containing the TTF-1 binding
site (
187) showed the formation of two protein-DNA complexes
(Fig. 4). Formation of these
complexes was abolished in the presence of molar excess of wild-type
but not of mutant oligonucleotide. Furthermore, excess SP-B
promoter oligonucleotide containing TTF-1 binding sites at
112
and
102 nucleotides but not mutant oligonucleotide abolished formation of the complex. These data indicated that the formation of a
protein-DNA complex is the result of binding of TTF-1. Incubation of
nuclear extracts with a polyclonal antibody to TTF-1 resulted in a
further retardation of the DNA-protein complex, demonstrating the
identity of the binding protein as TTF-1.
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EMSA using a SP-B promoter oligonucleotide containing the Sp1
site (162) and H441 nuclear extract showed the formation of two
protein-DNA complexes. The formation of these complexes was abolished
in the presence of excess wild-type but not of mutant oligonucleotide.
Similarly, excess Sp1 consensus oligonucleotide but not mutant
oligonucleotide inhibited the formation of the complex. To identify
proteins binding to the Sp1 element, the effects of antibodies to
various members of the Sp1 family of proteins on the mobility of the
protein-DNA complex were investigated. Nuclear proteins were reacted
with antibodies before incubation with the labeled oligonucleotide, and
the protein-DNA complexes were analyzed by EMSA. Results showed that
Sp1 and Sp3 antibodies reacted with protein-DNA complexes to further
retard their mobility (Fig.
5), whereas Sp2 and Sp4
antibodies had no effect on the mobility of the protein-DNA complex
(data not shown). Specifically, complex 2 was abolished by the
Sp3 antibody, resulting in the formation of a supershifted complex.
These data suggested that a major component of complex 2 must
be Sp3. These results demonstrated the identity of proteins binding to
the Sp1 DNA element as Sp1 and Sp3 proteins. In a previous study,
Margana and Boggaram (20) found that the protein-DNA complexes formed
by Sp1 elements in the SP-B promoter were supershifted by
Sp1 and Sp3 antibodies but not by Sp2 and Sp4 antibodies.
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EMSA using the SP-B promoter oligonucleotide containing the Ets
site (149) produced a minor band that was not supershifted by
Ets antibodies (data not shown). The Ets site is overlapped by a
sequence that has similarities to NF-
B binding element, and the DNA
binding activity of the NF-
B element was strongly induced by
treatment with tumor necrosis factor-
(25 ng/ml) (data not shown).
The identity of the protein(s) present in untreated cells that
interacts with the Ets/NF-
B site remains to be investigated.
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DISCUSSION |
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SP-B mRNA is expressed in a highly cell-restricted manner by alveolar
type II and bronchiolar (Clara) epithelial cells of the lung (24, 34)
and is developmentally induced (18). Human (4, 33) and rabbit (19)
SP-B promoter fragments containing the 218 to +41 and
236 to +39 nucleotides support high-level expression of CAT
activity in NCI-H441 cells. SP-B promoter serves as a target
for TTF-1 and HNF-3/forkhead transcription factors that bind and
activate SP-B promoter function (3, 9, 20). Margana and
Boggaram (19) previously found, by deletion mapping, that a region of
rabbit SP-B gene comprising the
236 to +39 nucleotides is necessary and sufficient for high-level promoter activity in NCI-H441 cells. Deletion of the 5' region of the rabbit
SP-B minimal promoter to
140 nucleotides reduced
promoter activity by nearly 80% (19). A previous study by Margana and
Boggaram (20) showed that Sp1, Sp3, TTF-1, and HNF-3 transcription
factors bind to sequence motifs within the
140 nucleotide to
activate the promoter. These data suggested that the upstream region of
the rabbit SP-B promoter,
236 to
140 nucleotides, is
essential for promoter function and contains functionally important
cis-DNA elements.
Identification of cis-DNA elements and interacting
transcription factors important for SP-B promoter activity is
essential for the understanding of mechanisms underlying cell
type-specific and developmental regulation of SP-B gene
transcription. In the present study, we mapped the SP-B
upstream promoter region for the identification of functionally
important regions and identified cis-DNA elements and
interacting transcription factors important for SP-B promoter
activity. Deletion of 5'-flanking DNA of SP-B minimal
promoter to the 170 and
140 nucleotides reduced
promoter activity by 40 and 80%, respectively, indicating that the
236 to
170 and
170 to
140 nucleotide
regions are essential for promoter activity. A previous study (19)
showed that a region derived from the SP-B minimal promoter,
236 to
39 nucleotides, increased CAT expression
from SV40 minimal promoter in a lung cell-specific manner in
cell cultures. We further examined SP-B sequences
236 to
140 and
140 to
27 for enhancing activity. Both
regions increased CAT expression from SV40 minimal promoter by
a similar magnitude, indicating that they contained enhancing activities. These data indicated that the upstream and proximal regions
of the promoter are required for the full expression of the activity of
the SP-B enhancer.
The SP-B upstream promoter region, 236 to
140
nucleotides, is essential for promoter activity and contains
significant enhancing activity, indicating that it contains
functionally important cis-DNA elements. We analyzed the
functional importance of putative cis-DNA elements by
mutational analysis. cis-DNA elements were mutated to prevent
binding of transcription factors, and the effects of the mutations on
the promoter activity were determined. Mutational analysis of the
SP-B upstream promoter region revealed the presence of three
functionally important DNA elements with similarities to TTF-1, Sp1,
and Ets/NF-
B binding motifs. Mutation of TTF-1 (
187 to
182 nucleotides), Sp1 (
162 to
154 nucleotides), or Ets/NF-
B (
149 to
146 nucleotides) elements reduced
promoter activity by ~50%. Margana and Boggaram's previous study
(20) demonstrated that sequence elements with similarities to Sp1 and Ets motifs at
207 and
162 nucleotides did not have
functional roles in SP-B promoter activity. The results of
mutational analysis are in agreement with the deletion analysis of the
SP-B upstream region. Deletion of 5' region of
SP-B promoter to the
170 nucleotide removes the TTF-1
binding sequence and consequently reduces promoter activity by ~50%.
Similarly, further deletion to the
140 nucleotide removes Sp1
and Ets/NF-
B sequences and further decreases promoter activity by
~50%.
EMSA using TTF-1 antibodies resulted in further retardation of the
protein-DNA complex identifying TTF-1 as the major protein factor
binding to the TTF-1 element at the 187 nucleotide. In a
previous study, Margana and Boggaram (20) identified that TTF-1 binding
sites at
112 and
102 nucleotides serve important roles in
SP-B promoter function. TTF-1, a homeodomain-containing transcription factor, plays important roles in the regulation of
thyroid-specific gene expression (13). Apart from its expression in the
thyroid and certain regions of the fetal brain, TTF-1 is also expressed
in the lung. TTF-1 is selectively expressed in alveolar type II cells
and subsets of bronchiolar epithelial cells in the lung and functions
as a transcriptional activator of SP-A (6), SP-B, and
SP-C and Clara cell secretory protein (CCSP) genes (3).
Of the two putative Sp1 elements, the element at the
162 nucleotide was found to be important for promoter
activity and binding Sp1 and Sp3 transcription factors. Margana and
Boggaram (20) found previously that Sp1 and Sp3 proteins bind to
functional Sp1 elements at
130 and
35 nucleotides in the
SP-B promoter. Because all members of the Sp family
of transcription factors display overlapping binding specificity and
affinities, it is likely that lack of binding of Sp2 and Sp4 proteins
to the Sp1 element is due to the absence of the proteins in NCI-H441
cells rather than to their inability to recognize the binding sequence. The Sp1 element (
162 site) contained an overlapping sequence with similarities to the Ets binding motif. The previous study (20)
showed that mutation of the Ets motif at the
162 site had
no effect on promoter activity, and proteins that bound to the
Ets element were not recognized by Ets antibodies.
The identity of proteins binding to the putative Ets element
(149 nucleotide) is not clear. The Ets element overlapped with a
NF-
B binding site and reacted weakly with nuclear proteins from
untreated cells. The protein-DNA complex was not recognized by Ets
antibodies. It remains to be investigated whether the Rel family of
proteins that bind to the Ets/NF-
B element in nuclear extracts from
tumor necrosis factor-
-treated cells plays an important role in
SP-B promoter function.
Results of the present study and a previous study by Margana and
Boggaram (20) have shown that the SP-B minimal promoter contains functional binding sites for Sp1, Sp3, TTF-1, and HNF-3 transcription factors (Fig. 6). The
previous study (20) also indicated that combinatorial or cooperative
interactions between transcription factors are necessary for promoter
function. During fetal lung development, TTF-1 and HNF-3 are expressed
before differentiation of alveolar type II cells and expression of SP-B
mRNA (36), suggesting that additional factors may be required for
activation of SP-B gene transcription. Our data indicate that
Sp1 and Sp3 serve as additional factors essential for the activation of
SP-B gene expression. The present and a previous study (20)
have demonstrated the importance of Sp1 and Sp3 transcription factors in the function of SP-B promoter. Sp1 and Sp3, which belong to a family of transcription factors that recognize GC boxes and contain
zinc finger motifs, also serve as important transcriptional activators
for other genes that are expressed in the respiratory epithelium, such
as 10-kDa Clara cell secretory protein (31), uteroglobin (11),
ClC-2 chloride channel (7), and the alveolar type I cell gene
T1 (26).
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TTF-1 and Sp1 serve as targets for phosphorylation by cAMP-dependent protein kinase and could potentially mediate the actions of hormones and other regulatory agents on SP gene expression in the developing lung. cAMP-dependent protein kinase A phosphorylation of TTF-1 has been implicated in the activation of SP-B (35) and SP-A (17) gene expression in lung epithelial cells. Sp1 has been shown to undergo phosphorylation by cAMP-dependent protein kinase A in HL60 leukemia cells (27). Whether phosphorylation of Sp1 by cAMP or other hormone-activated protein kinases controls SP-B gene expression in pulmonary epithelial cells is not known.
Our studies have identified multiple binding sites for TTF-1, Sp1, and Sp3 transcription factors in the SP-B promoter (Fig. 6). The clustered organization of the transcription factor binding sites in the promoter and their close proximity to the TATA element may promote cooperative interactions among the factors and facilitate stabilization of the transcriptional complex that may be required for activation of SP-B gene transcription. Our studies have also revealed that the SP-B promoter is a target for multiple transcription factors, some of which are expressed more selectively (such as TTF-1 and HNF-3) than others (such as Sp1 and Sp3). Interactions between multiple transcription factors and the general transcriptional complex on the SP-B promoter could give rise to the formation a stereospecific transcription complex that is essential for cell-type specific activation of SP-B gene transcription.
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ACKNOWLEDGEMENTS |
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This research was supported by National Heart, Lung, and Blood Institute Grant HL-48048.
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FOOTNOTES |
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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 and other correspondence: V. Boggaram, Dept. of Molecular Biology, The Univ. of Texas Health Science Center at Tyler, 11937, US Highway 271, Tyler, TX 75708-3154 (E-mail: vijay{at}uthct.edu).
Received 18 August 1999; accepted in final form 5 October 1999.
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