Departments of 1 Biochemistry
and 3 Obstetrics-Gynecology and
4 The Cecil H. and Ida Green
Center for Reproductive Biology Sciences, Surfactant
protein (SP) A gene transcription is developmentally regulated and
stimulated by hormones and factors that increase intracellular cAMP.
The baboon (b) genome contains two highly similar
SP-A genes,
bSP-A1 and
bSP-A2. With the use of a ribonuclease protection assay with gene-specific probes, the two bSP-A
genes were found to be differentially regulated during baboon fetal lung development in that expression of the
bSP-A2 gene appeared to be induced to
a high level at a later time in gestation than that of the
bSP-A1 gene. Both the
bSP-A1 and
bSP-A2 genes were found to be highly
responsive to the inductive effects of cAMP in baboon fetal lung
explants in culture. By DNase I footprinting and electrophoretic
mobility shift assays with bacterially expressed thyroid transcription
factor-1 (TTF-1) and type II cell nuclear extracts, three TTF-1 binding
elements were identified within the 255-bp region flanking the
5'-end of each bSP-A gene;
however, these differed in position and spacing for the two
bSP-A genes. To functionally define
the genomic regions that are required for cAMP regulation of
bSP-A gene expression in type II
cells, fusion genes composed of various amounts of 5'-flanking
DNA from the bSP-A1 and
bSP-A2 genes linked to the human
growth hormone structural gene as a reporter were transfected into type
II cells in primary culture. We found that 255 bp of 5'-flanking
DNA, which contain three TTF-1 binding elements, from
bSP-A1 and
bSP-A2 genes were sufficient to
mediate high basal and cAMP-inducible expression in type II cells. We
also observed that there were no obvious differences in the magnitude
of the responses of these fusion genes to cAMP treatment.
surfactant protein A; deoxyribonucleic acid; fetal lung; thyroid
transcription factor-1; ribonuclease protection assay
PULMONARY SURFACTANT, a lipoprotein synthesized
exclusively by alveolar type II cells, acts to reduce alveolar surface
tension, thereby preventing alveolar collapse on the exhalation of air. Four lung-specific proteins have been found to be associated with surfactant: surfactant protein (SP) A, SP-B, SP-C, and SP-D. These appear to serve differential roles in the reduction of alveolar surface
tension, surfactant phospholipid reutilization, and immune defense
within the alveolus (15).
Expression of the gene encoding SP-A, the major surfactant protein, is
lung specific; SP-A is expressed primarily in alveolar type II cells
and, to a lesser extent, in bronchiolar epithelial (Clara) cells (3,
38). Expression of the SP-A gene is
also developmentally regulated; gene transcription is initiated in the
fetal lung only after ~70% of gestation is completed and reaches maximum levels toward term (5). Various hormonal factors have been
found to regulate SP-A gene
expression; cAMP and glucocorticoids are reported to have major
regulatory effects (24, 28, 31, 32). In studies using
rabbit (29), human (32), and baboon (35) fetal lungs in organ culture,
SP-A mRNA and protein levels were found to be augmented by cAMP analogs
and by agents that increase the levels of intracellular cAMP. cAMP also
enhances the rate of type II cell differentiation and enlargement of
prealveolar ducts (32). The effects of glucocorticoids on
SP-A gene expression are more complex;
e.g., in human fetal lung explants, dexamethasone (Dex) acts
synergistically with cAMP to increase
SP-A gene transcription. However, Dex
causes a dose-dependent decrease in SP-A mRNA stability (6, 7).
SP-A is encoded by a single-copy gene in rabbits (5), rats (11), dogs
(4), and mice (19); however, it has been found that the human (16, 26)
and the baboon (13) genomes contain two highly similar
SP-A genes
(SP-A1 and
SP-A2). The two human (h)
SP-A genes
(hSP-A1 and
hSP-A2) appear to be differentially regulated during development and by cAMP and glucocorticoids (27). In
lung tissue from a 28-wk-gestation neonate, only hSP-A1 mRNA transcripts were detected, whereas in adult human lung tissues, the
ratio of hSP-A2 to hSP-A1 mRNA transcripts was found to be 3:1 (27).
These preliminary findings suggest that expression of hSP-A1 is
initiated earlier in development than that of hSP-A2, whereas hSP-A2
transcripts are more highly expressed postnatally. The
hSP-A2 gene also was found to be more
responsive to the inductive effects of cAMP than the
hSP-A1 gene (27). In midgestation
human fetal lung explants cultured in control medium, the
majority of SP-A transcripts were found to be SP-A1; in tissues
cultured in the presence of dibutyryl cAMP (DBcAMP), the ratio of
SP-A2 to SP-A1 mRNA was increased to levels similar to that in adult
lung tissues (27).
Recently, Seidner et al. (35) reported the use of the baboon as a model
for study of SP-A gene regulation. In
studies using lung tissues of 92- to 140-day gestational age fetal
baboons (term 184 days), the effects of cAMP and Dex were highly
similar to those observed with lung explants of midgestation human
abortuses; i.e., cAMP had a marked stimulatory effect, whereas Dex
caused a dose-dependent decrease in SP-A mRNA levels (35). Sequence comparison of DNA upstream of the transcription initiation sites and
within the 3'-untranslated regions of the two baboon (b)
SP-A (bSP-A1 and
bSP-A2) and the two
hSP-A genes indicates that
bSP-A1 is more similar to
hSP-A1, whereas
bSP-A2 is more similar to
hSP-A2 (13).
In consideration of the similarities of the
hSP-A and
bSP-A genes and their hormonal
regulation, as well as of the limited availability of third-trimester
human fetal lung tissues, in the present study, we used the baboon as a
model to study differential regulation of the
SP-A1 and
SP-A2 genes during development and by
cAMP. To begin to understand the mechanisms whereby these two bSP-A genes are differentially
regulated, we transfected type II cells in primary culture with fusion
genes composed of up to 1,000 bp of 5'-flanking DNA from each
bSP-A gene to compare their capacity
to mediate cAMP induction of SP-A promoter activity. We also analyzed
the proximal 5'-flanking regions of both
bSP-A genes for binding sites for
thyroid transcription factor-1 (TTF-1), a homeodomain transcription
factor expressed specifically in the developing thyroid and lung
epithelia and in restricted areas of the developing brain (14, 21).
TTF-1 is required for organogenesis of the thyroid, lung, and anterior
pituitary (18) and mediates expression of thyroid-specific genes (10,
12, 14, 36). It has been reported that TTF-1 binds to and
transactivates the SP-A, SP-B, SP-C, and Clara cell-specific protein
(Clara cell 10-kDa secretory protein) gene promoters (8, 9, 17, 33). Recently, Li et al. (23) found that cAMP-responsive expression of the
bSP-A gene is mediated by increased
phosphorylation and binding of TTF-1.
Culture of fetal lung explants, isolation of type II
pneumonocytes, and preparation of type II cell nuclear
extracts. Fetal baboons were delivered by hysterotomy
after time-dated pregnancies that were confirmed by fetal morphometrics
on maternal ultrasounds at 70 and 100 days gestational age. All studies
were approved by the Institutional Animal Care Committee at the
Southwest Foundation for Biomedical Research (Dallas, TX) and strictly
adhered to the National Research Council's Guide for
Care and Use of Laboratory Animals. Fetuses were
delivered at the following gestational ages: 92, 125, 140, 160, and 175 days. Lung tissues were minced into 1- to
2-mm3 fragments and cultured on
lens papers supported by stainless steel grids in serum-free Waymouth
MB 752/1 medium alone or in medium containing DBcAMP (1 mM) (35, 37).
The procedure used to isolate and maintain type II pneumonocytes in
primary culture has been previously described (2). Briefly, lung
tissues of midgestation human abortuses were maintained in organ
culture for 5 days in serum-free Waymouth MB 752/1 medium in the
presence of DBcAMP (1 mM) to promote type II cell differentiation (32). The explants were then incubated with collagenase (0.5 mg/ml) to
disperse the cells and were subsequently treated with DEAE-dextran, which selectively eliminates fibroblasts. The enriched type II cell
suspension was plated onto culture dishes that were coated with an
extracellular matrix from Madin-Darby canine kidney (MDCK) cells and
cultured in Waymouth MB 752/1 medium containing 1 mM DBcAMP for another
4 days (2). Nuclear extracts were prepared from these isolated type II
cells as previously described (22, 23).
RNase protection assay. A 150-bp
32P-labeled antisense RNA probe
for the bSP-A1 gene was transcribed
from +3,678 to +3,827 bp of bSP-A1
genomic DNA (13). A 190-bp
32P-labeled antisense RNA probe
for the bSP-A2 gene was transcribed from +3,644 to +3,833 bp of bSP-A2
genomic DNA (13). A 32P-labeled
18S rRNA antisense probe was generated by in vitro transcription of
pTRI RNA 18S (Ambion, Austin, TX). To detect the transcripts of the
bSP-A1 or
bSP-A2 genes, 2.5 µg of total RNA
were combined with 2 × 104
counts/min (cpm) of the bSP-A1 or bSP-A2 RNA probe. Each reaction mixture also contained 1 × 103 cpm of 18S rRNA probe; 2 µg
of unlabeled 18S rRNA probe were added to each reaction to ensure that
the probe was in molar excess of 18S rRNA. Reaction mixtures were
heated at 95°C for 4 min, followed by incubation at 45°C
overnight. Reaction mixtures were then digested with 200 µl of a
diluted RNase A-RNase T1 mixture (1:100 dilution; Ambion) and incubated
at 37°C for 30 min. Protected RNA probes were then ethanol
precipitated, resolved on a 6% denaturing polyacrylamide gel, and
detected by autoradiography. The relative amounts of bSP-A mRNA and 18S
rRNA were assessed by scanning densitometry. After densitometry, the
levels of bSP-A1 and bSP-A2 mRNA transcripts were corrected by
normalization to the levels of 18S rRNA.
DNase I footprinting. A 321-bp TTF-1
cDNA fragment containing the entire homeodomain region
(TTFHD) was amplified with two primers (5'-TTFHD,
5'-TCCGACGTGAGCAAGAACATG-3' and
3'-TTFHD,
5'-TCACTGCTGCGCCGCCTTGTC-3'), with the baboon TTF-1 cDNA
clone as a template. This DNA fragment was incorporated into the
bacterial expression vector pGEX-KG (Pharmacia) in-frame with
glutathione S-transferase (GST). The GST-TTFHD polypeptide was prepared
from Escherichia coli according to
procedures suggested by the manufacturer (Pharmacia). DNA probes for
footprinting analysis were prepared by PCR with
32P end-labeled synthetic
oligonucleotides as primers (34). The bSP-A1 and
bSP-A2 genomic clones were used as
templates for the amplification of sequences between base pairs
Electrophoretic mobility shift assays.
Oligonucleotides were end labeled with T4 polynucleotide kinase and
[ Construction of bSP-A-human growth hormone fusion
genes and preparation of recombinant adenoviruses.
Fusion genes composed of 50, 253 or 255, and 1,068 or 986 bp of DNA
flanking the 5'-ends plus 40 bp of the first exons of the
bSP-A1 or
bSP-A2 gene linked to the human growth
hormone (hGH) structural gene as a
reporter were constructed. bSP-A1 and
bSP-A2 genomic sequences were
amplified from bSP-A1 and
bSP-A2 genomic clones (13) by PCR with
specific oligonucleotides containing sequences complementary to the
5'- and 3'-ends of the genomic regions to be amplified with
Hind III restriction sites near their
5'-ends and BamH I sites near
their 3'-ends. PCR was accomplished with
Taq polymerase (Boehringer Mannheim)
and a DNA thermal cycler (Perkin-Elmer Cetus) with protocols suggested
by the manufacturer. PCR fragments were digested with Hind III or
BamH I and subcloned into
pACsk20GH, which contains the left
17% of the human adenovirus 5 genome and the promoterless hGH
structural gene.
To generate recombinant adenoviruses, 293 cells, a permissive human
embryonic kidney cell line, were cotransfected with the fusion genes
and pJM17, which contains the entire adenovirus genome plus insertion
of a 4.3-kb plasmid. pJM17 itself is too large to be packaged into
viral particles. Infectious viral particles are formed on in vivo
recombination of the plasmids to produce a recombinant viral genome of
packageable size. Viral DNA was analyzed for the presence of the fusion
genes by restriction endonuclease digestion and DNA sequencing. The
number of infectious recombinant viruses were titered with 293 cells at
least twice to ensure the accuracy of the titer.
Expression of SP-A fusion genes in transfected type II
cells. Type II cells plated at a density of 5-9 × 106 cells/60-mm dish were
maintained overnight in Waymouth MB 752/1 medium containing 10% fetal
bovine serum. The cells were then washed twice with medium and
incubated for 1 h with 1 × 106 recombinant viral particles,
resulting in a multiplicity of infection of 0.1-0.2. In this
manner, the same number of cells (1 × 106) was infected in each
experiment. The medium was then aspirated and replaced with fresh
medium in the absence or presence of DBcAMP (1 mM). Medium from
transfected cells was collected every 24 h and assayed for hGH by RIA
(Nichols Institute).
Developmental changes in bSP-A1 and bSP-A2 gene
expression in the baboon fetal lung. In an initial
study by McCormick and Mendelson (27) of the regulation of
hSP-A gene expression, it was observed
that the hSP-A1 and
SP-A2 genes are differentially regulated during development and by cAMP. To determine whether the two
bSP-A genes are also differentially
regulated as their human counterparts, RNase protection assays were
used to analyze the expression of bSP-A1 and bSP-A2 mRNA transcripts at
different developmental stages. Two antisense RNA probes to regions of
the bSP-A genes that differ between
bSP-A1 and
bSP-A2 were generated by in vitro
transcription of their respective DNAs (lane
1, top bands, in both
Figs. 1 and 2).
An 18S rRNA probe was simultaneously used as a standard for the
normalization of loading. Radiolabeled probes were annealed with total
RNA from the lung tissues of fetal baboons of 92, 125, 140, 160, and
175 days gestational age (term 184 days), digested with RNase, and
fractionated by denaturing polyacrylamide gel electrophoresis. To
evaluate the specificity of these probes, sense RNA fragments of either
bSP-A1 or bSP-A2 were also transcribed from their respective DNAs and
analyzed for their ability to protect the antisense RNA probes from
RNase digestion. As expected, the bSP-A1 probe was protected from RNase digestion only by bSP-A1 RNA (Fig.
1A, lanes
3 and 4), whereas the bSP-A2 probe was protected from RNase digestion only by bSP-A2 RNA
(Fig. 2A, lanes
3 and 4). This
confirmed that each probe was specific for its respective mRNA. The
protected sense probes were somewhat longer than the protected bSP-A1
and bSP-A2 mRNAs in the tissue samples because the sense probes
contained 5' and 3' extensions that hybridized to part of
the 3' and 5' extensions of the antisense probes. The
expression levels of both bSP-A1 and bSP-A2 were analyzed by scanning
densitometry and were normalized to 18S rRNA (Figs.
1B and
2B). As can be seen, both bSP-A1 and bSP-A2 mRNA levels were barely detectable through 140 days gestational age but were markedly increased by day
175. At 160 days gestational age, bSP-A1 mRNA
transcripts were increased to levels comparable to those on
day 175, whereas bSP-A2 mRNA
transcripts were much lower compared with those on day
175. Reproducible findings were obtained with RNA
samples from an independent gestational series of baboon fetal lungs.
Therefore, expression of the bSP-A2
gene appears to be induced later during fetal development than that of
the bSP-A1 gene.
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
253 and +40 for bSP-A1 and
255 and +40 for bSP-A2. The
DNase I footprinting assays were performed in a 200-µl reaction
volume. DNA-binding reactions were carried out in a mixture containing
10 mM Tris · HCl (pH 8.0), 5 mM
MgCl2, 1 mM
CaCl2, 2 mM dithiothreitol, 50 µg/ml of BSA, 2 µg/ml of calf thymus DNA, and 100 mM KCl.
Bacterially expressed proteins (either GST or
GST-TTFHD peptide) were incubated
with 20,000 cpm of radiolabeled DNA fragment. After 30 min of
incubation at room temperature, the reaction mixtures were digested
with DNase I for 2 min and rapidly stopped by the addition of 700 µl of DNase I stop solution containing 645 µl of 100% ethanol, 5 µg
of tRNA, and 50 µl of saturated ammonium acetate. The reaction products were fractionated on 6% polyacrylamide-7 M urea sequencing gels; the sequencing of the probe was performed as described by Maxam
and Gilbert (25), and a sample of the sequencing reaction was
loaded adjacent to the samples analyzed by DNase I footprinting.
-32P]ATP.
Bacterially expressed GST-TTFHD
polypeptide (2 µg) or alveolar type II cell nuclear extracts (10 µg) were incubated at room temperature for 30 min in binding buffer
(20 mM HEPES, pH 7.6, 100 mM KCl, 0.2 mM EDTA, 0.5 mM dithiothreitol,
and 20% glycerol) with a radiolabeled DNA probe (10,000 cpm); 2 µg
of poly(dI-dC) · poly(dI-dC) were simultaneously
added as a nonspecific competitor. Protein-DNA complexes were resolved
on 5% nondenaturing polyacrylamide gels and visualized by
autoradiography. For DNA competition electrophoretic mobility shift
assay (EMSA), nonradiolabeled double-stranded oligonucleotides were
added simultaneously with labeled probe. Supershift EMSA was performed
by adding 1 µl of antiserum to the binding reaction, followed by a
30-min incubation at room temperature before electrophoresis.
RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
View larger version (30K):
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Fig. 1.
Developmental changes in baboon surfactant protein A1
(bSP-A1) gene expression.
A:
32P-labeled antisense RNA probe
specific for bSP-A1 gene was
transcribed from +3,678 to +3,827 bp of
bSP-A1 genomic DNA
(lane 1) (13). A
32P-labeled 18S rRNA probe was
generated by in vitro transcription of pTRI RNA 18S. Radiolabeled
probes were annealed with either 2.5 µg of yeast RNA
(lane 2), 0.1 µg of sense RNA
probes for bSP-A1 and bSP-A2
(bSP-A1sense and
bSP-A2sense, respectively)
generated by in vitro transcription of the corresponding genomic
sequences (lanes 3 and
4, respectively), or total RNA
isolated from lung tissues of 92 (d92)-, 125 (d125)-, 140 (d140)-, 160 (d160)-, and 175 (d175)-day gestational age fetal baboons
(lanes
5-9,
respectively). After RNase treatment and denaturing polyacrylamide gel
electrophoresis, radiolabeled RNA fragments were detected by
autoradiography. B: autoradiogram was
analyzed by scanning densitometry. Shown are arbitrary units for bSP-A1
mRNA that were normalized to 18S rRNA (bSP-A1/18S).
View larger version (27K):
[in a new window]
Fig. 2.
Developmental changes in bSP-A2 gene
expression. A:
32P-labeled antisense RNA probe
specific for bSP-A2 gene was
transcribed from +3,644 to +3,833 bp of
bSP-A2 genomic DNA
(lane 1) (13). A
32P-labeled 18S rRNA probe was
generated by in vitro transcription of pTRI RNA 18S. Radiolabeled
probes were annealed with either 2.5 µg of yeast RNA
(lane 2), 0.1 µg of
bSP-A1sense and
bSP-A2sense generated by in vitro
transcription of the corresponding genomic sequences
(lanes 3 and
4, respectively), or total RNA
isolated from lung tissues of 92-, 125-, 140-, 160-, and 175-day
gestational age fetal baboons (lanes
5-9,
respectively). After RNase treatment and denaturing polyacrylamide gel
electrophoresis, radiolabeled RNA fragments were detected by
autoradiography. B: autoradiogram was
analyzed by scanning densitometry. Shown are arbitrary units for bSP-A2
mRNA that were normalized to 18S rRNA (bSP-A2/18S).
Effects of DBcAMP on levels of bSP-A1 and bSP-A2 mRNA transcripts in cultured baboon fetal lung tissues. Previously, McCormick and Mendelson (27) observed that the hSP-A2 gene is far more responsive to the inductive effects of cAMP than the hSP-A1 gene. To analyze the effects of cAMP on bSP-A1 and bSP-A2 gene expression, lung tissues from 125-day gestational age fetal baboons were cultured in the absence (control) and presence of 1 mM DBcAMP and analyzed for bSP-A1 and bSP-A2 mRNA transcripts by RNase protection. Total RNA was isolated from baboon fetal lung tissues after 1, 3, or 5 days in culture. The RNA was annealed with either the bSP-A1 or bSP-A2 probe, digested with RNase, and fractionated by denaturing polyacrylamide gel electrophoresis. As shown in Fig. 3, levels of expression of both the bSP-A1 and bSP-A2 genes were markedly induced by DBcAMP after 5 days in culture; however, in this experiment, the time course for this response differed in that the bSP-A1 gene was induced to relatively high expression levels by DBcAMP after 1 day in culture, whereas a major effect of DBcAMP to increase bSP-A2 gene expression was not evident until day 5 in culture. In three independent experiments with fetal lung tissues from 125-day gestational age fetal baboons, both the bSP-A1 and bSP-A2 genes were found to be comparably induced by DBcAMP after 5 days in culture. On the other hand, the time courses of induction of the two bSP-A genes in response to cAMP were not consistent in all experiments; in one of the three experiments, bSP-A2 expression was also markedly induced by DBcAMP as early as day 1 of culture.
|
Sequence analysis of the 5'-flanking regions of
the bSP-A1 and bSP-A2 genes. To identify potential
cis-acting elements that regulate
bSP-A gene expression in type II cells
and the inductive effect of cAMP, we analyzed ~1.2 kb of DNA flanking
the 5'-ends of the bSP-A1 and
bSP-A2 genes (13). An alignment of the
5'-flanking sequences of the two
bSP-A genes with the two
hSP-A genes is shown in Fig.
4. A putative nuclear receptor binding site [cAMP
response element for the SP-A promoter
(CRESP-A);
TGACCT], previously found to be functionally required for cAMP
induction of promoter activity of the rabbit
SP-A (30) and the
hSP-A2 (39) genes in transfected type
II cells, was present in the 5'-flanking regions of both bSP-A genes (from 246 to
239 bp in bSP-A1 and
248
to
241 bp in bSP-A2). A
GT-box element (GGGGTGGG), found to be essential for basal and cAMP
regulation of the hSP-A2 gene, was
perfectly conserved in the bSP-A1 gene
from
66 to
59 bp and was present as GGGGTGTG in the
bSP-A2 gene from
67 to
60 bp. As can be seen in Fig. 4,
the 5'-flanking sequence of the
bSP-A1 gene is more similar to that of
the hSP-A1 gene, whereas the 5'-flanking sequence of
the bSP-A2 gene is more similar to
that of the hSP-A2 gene. This is
exemplified by deletions just upstream of the
CRESP-A element in both the
bSP-A2 and
hSP-A2 5'-flanking sequences.
|
Characterization of TTF-1 binding sites within the
proximal 5'-flanking regions of the two bSP-A
genes. It has been recently reported that four TTF-1
binding sites are located between 231 to
168 bp within
the 5'-flanking region of the mouse
SP-A gene. To localize TTF-1 binding
sites in the proximal 5'-flanking regions of the
bSP-A1 and
bSP-A2 genes, DNase I footprinting
assays were carried out with a bacterially expressed
GST-TTFHD peptide. Bacterially expressed GST peptide was used as a control. As shown in Fig. 5, two distinct protected regions were found to be
present upstream of each gene; these are designated as
footprinted region 1 (FP1) and FP2 for
bSP-A1 and FP2 and FP3 for
bSP-A2. FP2 represents a homologous
region upstream of the two bSP-A
genes; however, FP2 within the 5'-flanking region of the
bSP-A1 gene is longer than its
bSP-A2 counterpart.
|
Sequence analysis of FP2 revealed that a conserved TTF-1 binding site (CTCAAG) was near its 5'-end. An oligonucleotide [TTF-1 binding element (TBE) 2a] containing this conserved TTF-1 binding site was synthesized and used as radiolabeled probe for EMSA (Fig. 6). As can be seen, radiolabeled TBE2a formed a strong complex with GST-TTFHD but not with GST. Coincubation with a 100- to 1,000-fold excess of a nonradiolabeled TTF-1 binding oligonucleotide [oligo(C)] from the thyroglobulin gene promoter effectively abolished complex formation as did an excess of nonradiolabeled TBE2a itself. On the other hand, an oligonucleotide in which the conserved TTF-1 binding sequence (CTCAAG) in TBE2a was mutated to CTGTGC (TBE2am) failed to compete for binding with the radiolabeled TBE2a probe. Similarly, a nonspecific oligonucleotide (5'-AGAGTGGGTGACCTTAGCCA-3') failed to affect complex formation. These findings indicate that the CTCAAG sequence represents a TTF-1 binding site present in both the bSP-A1 and bSP-A2 genes.
|
The DNase I footprinting analysis suggested the presence of another TTF-1 binding site within FP2. Based on a similarity to known TTF-1 binding sequences in other genes (9), examination of the 3' portion of FP2 revealed that the CTCTAG sequence within the bSP-A1 gene and the CCTAAG sequence within the bSP-A2 gene could possibly serve as additional TTF-1 binding sites. As shown in Fig. 7, radiolabeled oligonucleotides corresponding to the 3' portions of the FP2 region (B1-TBE2b and B2-TBE2b) formed complexes with GST-TTFHD. Excess amounts of nonradiolabeled B1-TBE2b and B2-TBE2b effectively competed with the corresponding labeled probes for binding to the GST-TTFHD peptide. When CTCTAG in B1-TBE2b was mutated to the sequence of nucleotides in its corresponding position in the bSP-A2 gene (CTCCAA) or when CCTAAG in B2-TBE2b was mutated to the sequence of nucleotides in its corresponding position in the bSP-A1 gene (CCTTGG), these two mutagenized oligonucleotides (B1-TBE2bm and B2-TBE2bm) could no longer compete with the radiolabeled wild-type probes for binding to the GST-TTFHD peptide. These findings suggest that these sequences represent additional TTF-1 binding sites within the FP2 regions of their corresponding genes. Interestingly, the spacing of the two TTF-1 binding sites within the FP2 regions are different. There are 14 nucleotides present between TBE2a and TBE2b of the bSP-A1 gene, whereas there are only 5 nucleotides present between TBE2a and TBE2b of the bSP-A2 gene; this explains why the FP2 upstream of the bSP-A1 gene is wider than that of the bSP-A2 gene. Using similar competition EMSA, we determined that the CTGGAG sequence in FP1 (named TBE1) and the TTGTAG sequence in FP3 (named TBE3) represent additional TBEs within the 5'-flanking regions of the bSP-A1 and bSP-A2 genes, respectively (data not shown).
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To further confirm that the TTF-1 binding sites identified with the bacterially expressed GST-TTFHD peptide were also able to bind native TTF-1 protein, alveolar type II cell nuclear extracts were used in EMSA analysis. As shown in Fig. 8A, type II cell nuclear extracts formed a strong complex with radiolabeled TBE2a oligonucleotide (lane 2). The binding of radiolabeled TBE2a was effectively competed by a 500× molar excess of nonradiolabeled FP1 but was unaffected by a nonradiolabeled oligonucleotide for the corresponding region of the bSP-A2 gene (B2-F1; Fig. 8A, lanes 3 and 4). This again indicates that FP1 is present only in the 5'-flanking region of the bSP-A1 gene. Both nonradiolabeled B1-FP2 and B2-FP2 effectively competed with the TBE2a probe for binding to type II cell nuclear extracts (Fig. 8A, lanes 5 and 6), indicating that both oligonucleotides contain TTF-1 binding sites. In Fig. 8A, lanes 7 and 8, nonradiolabeled FP3 effectively competed for binding, whereas an oligonucleotide for the corresponding region of the bSP-A1 gene (B1-F3) did not affect complex formation, confirming that FP3 is present only in the 5'-flanking region of the bSP-A2 gene. As shown in Fig. 8B, this protein-DNA complex was displaced when TTF-1 antiserum was included in the binding-reaction mixture, indicating that TTF-1 is present in the type II cell nuclear protein-DNA complex.
|
Functional localization of genomic sequences that
mediate basal and cAMP induction of bSP-A1 and bSP-A2 promoter activity in type II cells. To define the genomic regions
upstream of the bSP-A1 and
bSP-A2 genes that are required for
cAMP regulation of bSP-A gene
expression in type II cells, bSP-A-hGH
fusion genes composed of 50, 253 or 255, and 1,068 or 986 bp of DNA
flanking the 5'-ends of the
bSP-A1 or
bSP-A2 gene and 40 bp from the first exon, which encodes the 5'-untranslated region, linked to the hGH structural gene as a reporter were
constructed. These fusion genes were incorporated into the genome of
the replication-defective human adenovirus 5 for highly efficient and
reproducible DNA transfer by infection of rat fetal lung type II cells
in primary culture. Because the same number of infectious viral
particles containing each fusion gene was used in all experiments and
the number of infectious particles were limiting relative to the number
of cells (multiplicity of infection ~ 0.2), the same number of type
II cells was infected with each construct in each experiment. This resulted in highly reproducible and comparable data from one experiment to another. To analyze the effects of cAMP on fusion gene expression, infected type II cells were incubated in the absence and presence of
DBcAMP (1 mM); bSP-A promoter activities were analyzed by
radioimmunoassay of hGH protein secreted into the culture medium over
each 24-h period. As shown in Fig. 9,
expression of both 50-bp bSP-A-hGH fusion gene constructs was barely detectable and unaffected by cAMP
treatment. By contrast, the fusion genes containing 253 bp of
bSP-A1 or
255 bp of
bSP-A2 5'-flanking DNA,
respectively, were expressed at comparably elevated basal levels and
were induced approximately sevenfold by DBcAMP. The expression levels
of the
1,068-bp bSP-A1-hGH or
986-bp bSP-A2-hGH fusion gene
were greatly reduced compared with those of the
253-bp
bSP-A1-hGH or
255-bp bSP-A2-hGH fusion gene, suggesting the
presence of upstream inhibitory elements; however, the induction by
cAMP remained similar to that observed with the
253-bp
bSP-A1-hGH or
255-bp
bSP-A2-hGH fusion gene. Again, the
basal and cAMP-induced expression levels of the
1,068-bp
bSP-A1-hGH and
986-bp
bSP-A2-hGH fusion genes were found to
be comparable. These findings are consistent with the observation with
RNase protection assays that expression of the bSP-A1 and
bSP-A2 genes are highly induced by
cAMP (Fig. 3). In studies using the transfected type II cells, there
was no obvious difference in the time course of response of these
fusion genes to cAMP treatment (data not shown). This is in contrast to
the findings from RNase protection assays that the temporal effects of
cAMP on expression of the bSP-A1 and
bSP-A2 genes in cultured baboon fetal
lung appear to be different (Fig. 3).
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DISCUSSION |
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In the present study, the expression of bSP-A1 and bSP-A2 mRNA transcripts at different developmental stages was characterized with RNase protection assays. Consistent with previous findings for the two hSP-A genes (27), the two bSP-A genes also appear to be differentially regulated during development. Both bSP-A1 and bSP-A2 mRNA levels were found to be barely detectable through 140 days gestational age. At 160 days gestational age, bSP-A1 mRNA transcripts were increased to levels comparable to those on day 175, whereas bSP-A2 mRNA transcripts were considerably lower than those on day 175. Therefore, high levels of expression of the bSP-A2 gene occur later during development compared with those of the bSP-A1 gene. By Northern analysis, the levels of total SP-A mRNA transcripts on day 160 were found to be comparable to those on day 175 (35), suggesting that at 160 days of gestation, bSP-A1 mRNA comprises the dominant SP-A gene transcripts. This result is consistent with the previous finding (27) that, in lung tissue from a 28-wk-gestation neonate, hSP-A1 mRNA transcripts represented the predominant SP-A gene transcripts. From the findings of the present study, it is unclear as to whether there are subsequent changes in the level of one bSP-A gene transcript relative to the other at term or postnatally.
The mechanisms for the apparent differential regulation of the two bSP-A genes during development are unclear. Because both genes are likely to be expressed in the same cell types, they should be accessed by the same complement of transcription factors. Differential regulation of the two genes is possibly due to differences in the DNA elements that mediate their regulation. The 5'-flanking sequences of the two bSP-A genes were therefore sequenced for analysis of putative cis-acting elements based on a similarity to previously characterized transcription factor binding sites. CRESP-A and a GT box, two previously characterized elements important for mediating basal and cAMP-induced expression of rabbit (1, 30) and human (39, 40) SP-A promoter activity in type II cells, were found to be conserved in the 5'-flanking regions of both bSP-A genes. By a combination of DNase I footprinting and EMSA, three TTF-1 binding sites were characterized in the 5'-flanking region of each bSP-A gene. As is the case for the previously characterized TBEs within other SP gene promoters, sequences of the TBEs within the two bSP-A gene 5'-flanking sequences were found to be highly degenerate. The core sequences for TTF-1 binding in the two bSP-A genes include motifs of CTCAAG, CTCTAG, CTGGAG, CCTAAG, and TTGTAG. Interestingly, the position and spacing of these TTF-1 binding sites within the 5'-flanking sequences of the two bSP-A genes also are different. Only TBE2a is conserved between the two bSP-A genes. Within FP2, the spacing between TBE2a and TBE2b is wider in the bSP-A1 gene than in the bSP-A2 gene; this explains why FP2 upstream from bSP-A1 is longer than that of bSP-A2. The TBE in FP1 (TBE1) is present only in the bSP-A1 gene, whereas that in FP3 (TBE3) is present only in the bSP-A2 gene. Interestingly, TBE3 is adjacent to the previously characterized GT box, whereas TBE1 is close to the CRESP-A element. Whether this positioning could potentially facilitate protein-protein interactions and thereby contribute to differential regulation of the two bSP-A genes remains to be determined.
In studies using cultured lung explants from 125-day gestational age fetal baboons, both the bSP-A1 and bSP-A2 genes were found to be markedly induced by DBcAMP. This is in contrast to the finding by McCormick and Mendelson (27) that the hSP-A2 gene is far more responsive to the inductive effects of cAMP than the hSP-A1 gene. Because the sequences of the bSP-A1 and bSP-A2 genes are more similar to each other than are those of the hSP-A1 and hSP-A2 genes (13), it is likely that during evolution, divergence of the hSP-A1 gene resulted in a decrease in its responsiveness to cAMP.
To characterize the genomic sequences that mediate basal and
cAMP-induced expression of the bSP-A1
and bSP-A2 genes in type II cells, rat
fetal lung type II cells in primary monolayer culture were transfected
with fusion genes containing various amounts of 5'-flanking
sequences from the bSP-A1 and
bSP-A2 genes linked to the
hGH structural gene as a reporter. We
found that 253-bp bSP-A1 and 255-bp
bSP-A2 5'-flanking sequences
were sufficient to direct relatively high levels of basal expression in
primary cultures of type II cells cultured in the absence of cAMP;
expression was stimulated approximately sevenfold by the addition of
DBcAMP to the culture medium. Both basal and cAMP-induced
expression levels of 253-bp
bSP-A1-hGH and
255-bp
bSP-A2-hGH fusion genes were found to
be comparable. The time courses of induction of these fusion genes in
response to cAMP also were similar (data not shown), suggesting either
that the mechanisms for the apparent differential regulation of the two
bSP-A genes during development observed with RNase protection assays of
baboon fetal lung RNA are not active in this system or that the DNA
elements responsible for the differential regulation of the two
bSP-A genes are not present within the
fusion gene constructs. The 253- and 255-bp 5'-flanking regions
of bSP-A1 and
bSP-A2, respectively, contain the
previously characterized CRESP-A
element and the GT box; both of these elements have previously been
found to be required for cAMP induction of SP-A promoter activity in
transfected type II cells (30, 39). Interestingly, expression levels of
the bSP-A1- and
bSP-A2-hGH fusion genes containing
1,068-bp bSP-A1 or
986-bp bSP-A2 5'-flanking
sequences were reduced compared with the expression of fusion genes
containing
253- or
255-bp 5'-flanking DNA. A similar finding was also obtained in deletional analysis of the 5'-flanking region of hSP-A2
gene (39), suggesting the presence of putative inhibitory elements
within the upstream regions and their conservation among species. The
location of these inhibitory elements and their functional importance
in contributing to the tissue-specific and developmental regulation of
SP-A gene expression are unknown at
present. More detailed deletional mapping of the SP-A upstream sequences is necessary
for localization of these putative transcriptional silencers.
Recently, Li et al. (23) reported that cAMP-induced expression of the SP-A gene is mediated by protein kinase A-induced phosphorylation of TTF-1, resulting in its increased DNA-binding and transcriptional activity. In type II cell transfection studies, we observed that mutation of each TBE within the bSP-A2 5'-flanking region characterized in the present study caused a reduction in basal and cAMP-induced bSP-A2 promoter; mutation of TBE2a had the most deleterious effect (23). Although it is clear that the integrity of these TBEs is required for the maximal cAMP induction of SP-A promoter activity (23), the findings of the present study suggest that the differences in position and spacing of the TBEs between the bSP-A1 and bSP-A2 genes do not affect responsiveness to cAMP. We suggest that the cAMP-induced increase in TTF-1 phosphorylation and DNA-binding activity may provide the primary mechanism whereby cAMP induces SP-A gene expression, whereas differential regulation of expression of the bSP-A1 and bSP-A2 genes during development may involve other mechanisms, such as a unique cooperative interaction between transcription factors in the context of gene-specific differences in chromatin structure.
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ACKNOWLEDGEMENTS |
---|
We are grateful to Margaret Smith for expert help with cell and tissue culture.
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FOOTNOTES |
---|
This research was supported by National Heart, Lung, and Blood Institute (NHLBI) Grant U10-HL-52647. The baboon tissues were provided by the Bronchopulmonary Dysplasia Resource Center (San Antonio, TX) that was funded by NHLBI Grant HL-52636.
J. Li was supported by a predoctoral fellowship from the Chilton Foundation, Dallas, TX.
The nucleotide sequences reported in this paper have been submitted to GenBank with accession numbers AF061967 for baboon surfactant protein A1, AF061968 for baboon surfactant protein A2, and AF061969 for human surfactant protein A2 gene 5'-flanking sequences.
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. R. Mendelson, Dept. of Biochemistry, The Univ. of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd., Dallas, TX 75235-9038.
Received 26 May 1998; accepted in final form 21 August 1998.
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