Departments of 1 Biochemistry and 2 Obstetrics and Gynecology, The University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390-9038
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ABSTRACT |
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Expression of the pulmonary surfactant protein A (SP-A) gene is lung specific, developmentally regulated, and enhanced by hormones and factors that increase cAMP. We previously identified two E-box-like enhancers termed distal binding element (DBE) and proximal binding element (PBE) in the 5'-flanking region of the rabbit (r) SP-A gene that are essential for cAMP induction of rSP-A promoter activity (Gao E, Alcorn JL, and Mendelson CR. J Biol Chem 268: 19697-19709, 1993). We also found that DBE and PBE serve as binding sites for the basic helix-loop-helix-leucine zipper transcription factor, upstream stimulatory factor-1 (USF1) (Gao E, Wang Y, Alcorn JL, and Mendelson CR. J Biol Chem 272: 23398-23406, 1997). In the present study, PBE was used to screen a rabbit fetal lung cDNA expression library; a cDNA insert encoding the structurally related rabbit upstream stimulatory factor-2 (rUSF2) was isolated. The levels of rUSF2 mRNA reach peak levels in fetal rabbit lung at 28 days of gestation, in concert with the time of maximal induction of SP-A gene transcription. In yeast two-hybrid analysis, rUSF2 was found to preferentially form heterodimers, compared with homodimers, with rUSF1. Binding complexes of nuclear proteins isolated from fetal rabbit lung type II cells with the DBE and PBE were supershifted by anti-rUSF2 antibodies. Binding activity was enriched in nuclear proteins from type II cells compared with fibroblasts. Overexpression of rUSF2 in transfected lung A549 cells increased rSP-A promoter activity and acted synergistically with rUSF1. We suggest that heterodimers of USF2 and USF1 bound to two E-box elements in the SP-A gene 5'-flanking region serve a key role in developmental and hormonal regulation of SP-A gene expression in pulmonary type II cells.
upstream stimulatory factor; E-box; surfactant protein A; gene expression; development; type II cells; regulation
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INTRODUCTION |
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ADAPTATION OF THE
FETUS to extrauterine life is highly dependent on the maturity of
the lung at birth and its capacity to produce surfactant.
Surfactant, a developmentally regulated lipoprotein produced by
pulmonary type II cells, acts to reduce alveolar surface tension and
prevent atelectasis; its production is initiated in fetal lung only
after ~75% of gestation is completed. Lung surfactant contains at
least four associated proteins, surfactant protein (SP)-A, SP-B, SP-C,
and SP-D, which appear to serve important roles in surface activity,
phospholipid reutilization, and immune function within the alveolus
(19). The surfactant protein genes are developmentally
regulated and expressed in a lung-specific manner. The gene encoding
SP-A is expressed primarily in type II cells and to a lesser extent in
bronchioalveolar epithelial (Clara) cells (4, 37).
Transcription of the rabbit (r) SP-A gene is initiated in
fetal lung tissue on day 24 and reaches maximal levels
by day 28 (term = day 31) of
gestation. In studies using transgenic mice carrying fusion
genes comprising various amounts of 5'-flanking sequence from the
rSP-A gene linked to the human growth hormone
(hGH) structural gene, as reporter, our laboratory found
that as little as 378 bp of rSP-A 5'-flanking DNA were required to mediate appropriate lung tissue and cell-specific and
developmental regulation of reporter gene expression
(2).
SP-A gene expression in fetal lung is under multifactorial
control; agents that increase cAMP (26) and cytokines
(20) appear to play important roles in its regulation. In
studies using cultured rabbit fetal lung explants, our laboratory found
that cAMP causes a marked induction of rSP-A gene expression
(8, 25); the stimulatory effect of cAMP appears to be
mediated primarily at the transcriptional level (7). In
type II cell transfection studies to functionally define the genomic
regions that regulate basal and cAMP induction of rSP-A
promoter activity, our laboratory found that rSP-A
5'-flanking sequences between 47 and
378 bp are essential for cAMP
induction of SP-A promoter activity, whereas sequences
between
378 and
991 bp enhance overall levels of basal and
cAMP-induced expression (1).
Within the 378-bp SP-A 5'-flanking region, we have
identified a number of conserved response elements, of which each is
critical for cAMP induction of SP-A promoter activity. These
include a putative nuclear receptor half-site [cAMP response element
for the SP-A promoter (CRESP-A);
5'-TGACCTCA-3'] (28, 38), a thyroid transcription
factor-1 (TTF-1)-binding element (TBE; 5'-CTTCAAGG-3') (21), a GT box that binds Sp1 (5'-GGGGTGGGG-3')
(39), and an E-box-like sequence [proximal binding
element (PBE); 5'-CTCGTG-3'].
By use of electrophoretic mobility shift assays (EMSA); we observed
that the PBE (87 to
70 bp) competed for binding to rabbit lung
nuclear proteins with a structurally related element at
986 to
977
bp within the rSP-A 5'-flanking region, termed the distal binding element (DBE; CACGTG) (13). Binding activity for
DBE and PBE was found to be greatly enriched in nuclear extracts of lung type II cells compared with those of whole lung tissue
(13). In type II cell transfection studies to assess the
functional roles of the DBE and PBE in cAMP regulation of
SP-A promoter activity, we observed that
rSP-A
990:hGH fusion genes containing both the
DBE and PBE were induced ~30-fold by cAMP treatment
(13). The finding that mutation of the DBE
reduced basal and cAMP-induced expression to levels similar to those
found for promoter constructs containing
378 bp of rSP-A
5'-flanking DNA (15-fold induction by cAMP) suggests that the DBE
serves as a general, rather than as a specific, enhancer of
cAMP-regulated expression. On the other hand, mutagenesis of
the PBE [rSP-A
990PBE(
):hGH] caused a
marked reduction of basal expression and a loss of cAMP-stimulated expression to levels comparable to those of the basal promoter construct (13). It is apparent that the PBE
serves a more critical role in basal and cAMP regulation of
SP-A promoter activity than does the DBE. Whether this is
due to its proximity to the promoter, or its interaction with other
transcription factors bound to adjacent response elements, remains to
be determined. Interestingly, the PBE lies near a DNase I
hypersensitive site at approximately
100 bp, which was found to be
present in nuclei from rabbit lung several days before the time of
initiation of rSP-A gene transcription on day 24 but not in nuclei from liver or kidney tissues (10).
To characterize transcription factors that bind to these E-box motifs, radiolabeled PBE was used to screen a rabbit fetal lung cDNA expression library; cDNA inserts were isolated encoding two alternatively spliced forms of the basic-helix-loop-helix-leucine zipper (bHLH-LZ) transcription factor upstream stimulatory factor-1 (rUSF1a and b) (14). We found that USF1 gene expression is developmentally regulated in fetal rabbit lung and reaches a maximum at 23 days of gestation, just before the time of initiation of rSP-A gene transcription (14). Overexpression of rUSF1 in lung adenocarcinoma cells stimulated rSP-A promoter activity, suggesting that rUSF1 may serve a key role in the regulation of SP-A gene expression in pulmonary type II cells (14).
In the present study, a cDNA insert for another PBE-binding protein was characterized and found to encode rabbit upstream stimulatory factor-2 (rUSF2). rUSF2, which also is enriched in type II cells, was found to heterodimerize with rUSF1 in vivo and to act synergistically with rUSF1 to increase SP-A promoter activity. rUSF2 mRNA levels also were found to be developmentally regulated in fetal rabbit lung in concert with rSP-A gene transcription.
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EXPERIMENTAL PROCEDURES |
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Cloning of a cDNA insert encoding a PBE-binding protein.
First-strand cDNA was synthesized from poly(A)+ RNA
isolated from 24-day gestational age fetal rabbit lung tissues using
random hexanucleotides and was used to generate second-strand cDNA
using a cDNA synthesis kit (You-Prime cDNA synthesis kit, Pharmacia). The animal research protocols implemented in these studies were approved by the Institutional Animal Care and Use Advisory Committee of
the University of Texas Southwestern Medical Center at Dallas. The
double-stranded cDNAs with EcoRI/NotI linkers
were inserted into gt11 vector and packaged by use of Gigapack II
gold packaging extract (Stratagene). A 32P-labeled
double-stranded oligonucleotide corresponding to the PBE (core sequence
underlined) (5'-GAGGCCCTCGTGACAGGG-3') was used to screen
the
gt11 cDNA expression library employing standard techniques
(32). Approximately 4 million recombinant phage clones were screened, and two specific cDNA clones encoding proteins that
bound specifically to radiolabeled DBE
(5'-GATCTCCCACGTGGGTGCAGGG-3') and PBE, but not to
nonspecific C2 DNA (5'-TGCAGGGCCCAAGGACCTGGGCCATC-3') (13), were isolated. The cDNA inserts were subcloned into
the pGEM-7Z plasmid vector (Promega) and sequenced using Sequenase 2.0 (USB). One of the cDNA inserts was found to be highly similar to that
of human upstream stimulatory factor-1 (hUSF1), which has been reported
(14). The nucleic acid sequence of the other 1,640-bp cDNA
insert, termed pG-U2, was found to be highly similar to the sequence of
human upstream stimulatory factor-2 (hUSF2), but it lacked a
5'-untranslated region and 87 bp of coding sequence for the amino
terminus of the protein.
Construction of plasmids.
pGST-U2, used for expression of the glutathione
S-transferase (GST)-rUSF2 fusion protein in bacteria, was
constructed by cloning the EcoRI fragment of pG-U2 into the
EcoRI sites of pGEX-1T (Pharmacia) (Table
1). pGST-U230-228, which
lacks the bHLH-LZ region, was constructed by cloning the PCR-generated
cDNA fragment that encodes USF230-228 into
pGEX-1
T. This was used for expression of the GST-rUSF2
fusion protein in bacteria and for raising antibodies.
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Expression and purification of GST fusion proteins.
GST and GST fusion proteins were expressed and purified from
Escherichia coli DH-5 (Life Technologies) as described by
Smith and Johnson (35). After binding to
glutathione-Sepharose 4B (Pharmacia), the proteins were washed and
eluted with reduced glutathione (Sigma Chemical). The concentrations of
the expressed proteins were determined by the method of Bradford
(9) (Bio-Rad). The purity and sizes of the eluted proteins
were then evaluated by Coomassie blue staining of SDS-polyacrylamide gels.
Preparation of USF2 antibodies. Polyclonal antibodies to rUSF2 were generated by immunizing guinea pigs with GST-rUSF230-228, using methods described previously (18). The IgGs against GST were removed by passing the antiserum twice through a GST-agarose column. The antibodies produced recognized a protein band of 44 kDa, but they did not recognize GST or rUSF1 by Western blotting analysis. The immunoreactive band appears in some blots as a doublet, which may represent alternatively spliced forms or some type of posttranslational modification (data not shown).
EMSA.
Nuclear proteins were prepared (16) from type II cells and
fibroblasts isolated from cultured fetal rabbit lung explants (3). Binding reactions and gel electrophoresis were
performed as described previously (13). Double-stranded
oligonucleotides corresponding to the wild-type DBE and PBE and mutant
forms of DBE (5'-GATCTCCTACGTGGGTGCAGGG-3') and
PBE (5'-GAGGCCTTCGTGACAGGG-3'), in which the 5'
C in the E-box core sequence was mutated to T [underlined in core
sequence (bold)], and the canonical CRE (24) were
end-labeled using polynucleotide kinase and [-32P]ATP.
The nuclear extracts (10 µg) or bacterially expressed GST (240 ng) or
GST-rUSF2 fusion proteins (75 ng) were incubated at room temperature
for 20 min in binding buffer (20 mM HEPES, pH 7.6, 150 mM KCl, 0.2 mM
EDTA, 20% glycerol) with radiolabeled DNA probe and
poly(dI-dC)-poly(dI-dC) (Pharmacia) as nonspecific competitor, followed
by addition of guinea pig preimmune serum or the antibodies against
rUSF2 followed by another 20-min incubation. The DNA-protein complexes
were resolved on a 5% native polyacrylamide gel and visualized by autoradiography.
Dimerization of rUSF2 in a yeast two-hybrid system. To study the interaction of rUSF2 in vivo, the Gal4 DBD-USF2bHLH-LZ fusion plasmid, pDBD-U2, Gal4 activation domain-USF2bHLH-LZ fusion plasmid, pAD-U2, and the Gal4 DBD-USF1bHLH-LZ fusion plasmid, pDBD-U1, Gal4 activation domain-USF1bHLH-LZ fusion plasmid, pAD-U1 (14), as well as control plasmids pGBT9 (containing Gal4 DBD) and pGAD424 (containing Gal4 AD) were transformed individually into yeast HF7c (provided in the Matchmaker Two-Hybrid System kit, Clontech) or were cotransformed in different combinations (29). The transformants were grown in appropriate medium and studied as described previously (14).
Northern blot analysis of type II pneumonocyte and lung fibroblast mRNA. Total RNA was extracted from cells by homogenization in guanidinium isothiocyanate (4.0 M) using a Teflon-glass homogenizer. The cell extracts were centrifuged through a cesium chloride gradient (5.7 M), and the pelleted RNA was resuspended in water (11). Total RNA was electrophoresed, transferred to nitrocellulose, and probed using a 32P-labeled cDNA for rUSF2 using methods described in detail previously (8). Relative levels of mRNA were assessed by autoradiography.
RNase protection assay. RNase protection was used for analyzing developmental changes in USF2 mRNA levels because it is highly sensitive. Furthermore, 18S rRNA levels were determined simultaneously for correction of loading and transfer. A 107-bp 32P-labeled antisense RNA probe for rUSF2, corresponding to the region encoding amino acids 30-65 of rUSF2, was generated by in vitro transcription of the corresponding cDNA sequence. A 32P-labeled 18S probe was generated by in vitro transcription of pTRI RNA 18S (Ambion, Austin, TX). The radiolabeled probes were annealed with 2.5 µg of either yeast RNA or total RNA isolated from various gestational age fetal rabbit lung tissues and then were digested with RNase for 30 min. After ethanol precipitation, radiolabeled fragments were resolved on a 6% denaturing polyacrylamide gel and detected by autoradiography.
Transient transfections.
A549 lung adenocarcinoma cells (ATCC CCL 185) (22) were
plated at a density of 5-9 × 106 cells/60-mm
dish 1 day before transfection. The cells were cultured overnight in
Waymouth MB752/1 medium containing fetal calf serum (10%, vol/vol).
The cells were then washed three times with Hanks' balanced salt
solution (Life Technologies) and incubated with 11 µg of each DNA
fragment and 44 µg of Dotap (BMB) in Waymouth MB752/1 for 18 h.
The medium was then aspirated and replaced with fresh Waymouth MB752/1
at 24-h intervals. Two days later, the culture media and cells were
harvested. The concentrations of hGH in the media were analyzed by
radioimmunoassay using an Allegro hGH kit (Nichols Institute
Diagnostics, San Juan Capistrano, CA). Variations in transfection
efficiency were corrected by normalizing for -galactosidase
activity, which was assayed using a Galato-light kit (Tropix, Medford, MA).
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RESULTS |
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Isolation of the rUSF2 cDNA clone.
A cDNA isolated upon screening ~4 million recombinant phage clones
from a gt11 fetal rabbit lung cDNA expression library using
32P-labeled PBE as probe, encoded a protein that bound
specifically to radiolabeled PBE and DBE but not to nonspecific C2 DNA
(5'-TGCAGGGCCCAAGGACCTGGGCCATC-3') (13). The nucleic acid
sequence of the 1,640-bp cDNA insert, termed pG-U2, was found to be
highly similar to that of hUSF2 (34), but it lacked a
5'-untranslated region and 87 bp of sequence coding for the
NH2 terminus of the protein (GenBank accession no.
AY168774).
USF2 comprises one component of the complex of proteins bound to
the DBE and PBE.
To obtain expressed and purified rUSF2 protein for analysis of its
properties, the rUSF2 cDNA insert was subcloned into the GST fusion
vector pGEX-1T (Pharmacia). The GST-rUSF2 fusion proteins expressed
in E. coli and purified using glutathione-agarose beads were
used to analyze binding activity for the DBE and PBE by EMSA. As shown
in Fig. 1, nuclear proteins from 28-day
gestational age fetal rabbit lung (lanes 4, 8,
and 12) and GST-rUSF2 (lanes 3, 7, and
11) bound to the radiolabeled DBE (top) and PBE
(bottom). When in vitro translated rUSF2, which does not
contain GST, was used in EMSA, the same mobility shift complexes were
observed (data not shown). Although GST-rUSF2 fusion protein is ~15
kDa larger than rUSF2, its binding complexes with the DBE or PBE
displayed an electrophoretic mobility similar to that of native
rUSF2-containing complexes (e.g., lane 3 vs. lane
4), suggesting that folding and/or net charge of rUSF2 play
a more important role in mobility in native polyacrylamide gels than
does molecular mass. Similar findings were obtained when
GST-rUSF1 fusion proteins were used in EMSA (14). The
binding complexes of the DBE and PBE with fetal rabbit lung nuclear
proteins were supershifted by addition of anti-rUSF2 antibodies
(lane 12), indicating that the nuclear proteins that bound
to the DBE and PBE comprise USF2 homodimers and/or USF2 heterodimers.
The anti-rUSF2 antibodies also supershifted the complexes of expressed
GST-rUSF2 bound to the DBE and PBE (lane 11).
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USF2 DNA-binding activity is enriched in type II
pneumonocytes compared with lung fibroblasts.
The SP-A gene is expressed predominantly in type II
pneumonocytes and to a lesser extent in Clara cells of rabbit lung
tissue (4, 37). Binding activity of rabbit fetal lung
nuclear proteins (13) and of rUSF1 (14) for
the DBE and PBE is enriched in type II cells compared with whole lung
tissue and with lung fibroblasts isolated from the same
collagenase-digested cell suspension of cultured fetal rabbit lung
tissue. To evaluate the possible role of rUSF2 in the type II
cell-specific regulation of SP-A gene expression, EMSA was
used to assess the binding activity of rUSF2 in nuclear proteins from
an enriched population of the type II cells compared with nuclear
proteins from an enriched population of fibroblasts isolated from the
same collagenase-digested cell suspension of cultured fetal rabbit lung
tissue. As can be seen in Fig.
2A, binding activity for
radiolabeled DBE and PBE was greatly enriched in type II cells
(lane TII); essentially no binding activity was detected in
lung fibroblasts (lane Fb). The binding complexes of the DBE
and PBE with fetal rabbit lung type II cell nuclear proteins were
supershifted by addition of anti-rUSF2 antibodies, indicating that one
of the type II cell nuclear proteins that bound to the DBE and PBE was
rUSF2. To evaluate the integrity of the fibroblast and type II cell
nuclear extracts, we compared their abilities to bind a radiolabeled
double-stranded oligonucleotide containing the CRE sequence, TGACGTCA,
derived from rat somatostatin gene, which binds to the ubiquitous
transcription factor CRE binding protein (27). Binding
activities of nuclear proteins from fibroblast and type II cells for
the CRE were similar (data not shown), indicating that these two
preparations contained equivalent amounts of nuclear protein binding
activity. The absence of rUSF2 expression in lung fibroblasts was
further supported by Northern blot analysis, in which we obtained a
clear signal for rUSF2 mRNA in rabbit lung type II cells but were
unable to detect rUSF2 mRNA transcripts in lung fibroblasts isolated in
the same cell preparation (Fig. 2B).
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rUSF2 preferentially forms heterodimers with rUSF1 in vivo.
It has been reported that hUSF2 binds to an E-box motif as either
a homodimer or a heterodimer with USF1 (15, 34, 36). By
studying the interaction of 35S-labeled rUSF2 and rUSF1, as
well as bacterially expressed GST-rUSF2 and GST-rUSF1 fusion proteins
in the absence or presence of the DBE and PBE in vitro, we found that
rabbit USF2 also can form homodimers and heterodimers with rUSF1 in
solution in the absence of binding to the DBE and PBE and that binding
of rUSF2 to these E-boxes does not influence dimerization of rUSF2
(data not shown). A yeast two-hybrid system (5) was used
to determine whether homodimerization of rUSF2 and heterodimerization
of rUSF2 with rUSF1 occurs in vivo. As shown in Fig.
3, the bHLH-LZ region of rUSF2
homodimerized and, in turn, enhanced expression of the His3 gene in yeast HF7c (pDBD-U2/pAD-U2). The apparent efficiency of rUSF2
bHLH-LZ domains to form homodimers was only one third of that of the
bHLH-LZ domains of rUSF1. On the other hand, the efficiency of rUSF2
and rUSF1 bHLH-LZ domains to form heterodimers and consequently transactivate expression of the His3 gene in yeast HF7c was
fivefold greater than that of the bHLH-LZ regions of rUSF2 to form
homodimers and nearly twice as efficient as rUSF1 homodimerization.
These findings suggest that heterodimerization of rUSF2 and rUSF1
occurs with greater efficiency than does homodimerization and are in support of findings of others using alternative methodologies (36).
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rUSF2 mRNA levels are developmentally regulated in rabbit lung
tissue during development.
SP-A gene expression is developmentally regulated in fetal
lung tissue (26). In rabbits, SP-A gene
transcriptional activity is first detectable on day 24 and
reaches maximal levels by day 28 of the 31-day gestation
period (7). To analyze developmental changes in the levels
of rUSF2 mRNAs, aliquots of total RNA isolated from lung tissues of 21- to 28-day gestational age fetal rabbits and from neonates and adults
were analyzed by RNase protection assays. As shown in the autoradiogram
(Fig. 4A) and the accompanying graph of the scanned and corrected data (Fig. 4B), rUSF2
mRNA was present at comparable levels in fetal rabbit lung tissue on days 21 and 23 of gestation. rUSF2 mRNA
levels increased greater than twofold on day 25 of
gestation, reached maximal levels on day 28 and declined
markedly after birth. Similar findings were obtained using Northern
blot analysis of total RNA isolated from fetal rabbit lung tissue at
these different developmental stages. Again, relatively low levels of
rUSF2 mRNA transcripts were evident on days 21 and
23 of gestation with a marked induction on day 25 and further increase on day 28 (data not shown). This
developmental pattern is similar to that for SP-A gene
transcription rabbit lung tissue (7, 8).
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rUSF2 acts cooperatively with rUSF1 to increase SP-A:hGH fusion
gene expression in A549 cells.
In previous studies, we found that rSP-A:hGH fusion genes
containing 991 bp of rSP-A 5'-flanking sequence linked to
the hGH structural gene, as reporter
(rSP-A
991:hGH), were expressed in primary
cultures of type II cells and that their expression was markedly
stimulated by cAMP (1, 13). Mutagenesis of the DBE or PBE in the context of the 991 bp of 5'-flanking region resulted
in a pronounced reduction of basal and cAMP-induced fusion gene
expression (13). To determine the capacity of rUSF2 to regulate expression of the rSP-A gene via the DBE and PBE
and to act together with rUSF1, A549 cells were cotransfected with either SP-A
991:hGH,
SP-A
976:hGH (which lacks the DBE),
or SP-A
991PBE(
):hGH (containing a mutation in the PBE) alone or in combination with pCMV:USF2 and pCMV:USF1 expression vectors, added alone or in combination. A549 is a lung adenocarcinoma-derived cell line of presumed type II cell origin (22); however, these cells do not produce SP-A mRNA or
protein at detectable levels. Although the expressed rUSF2 lacked
29 amino acids from the NH2 terminus, we assumed
that this protein would have transcriptional activity comparable to the
full-length protein. In studies by Luo and Sawadogo (23),
it was found that a truncated form of USF2 lacking 40 amino acids from
the NH2 terminus had essentially full transcriptional
activity compared with the full-length protein. The transcriptional
activation and DNA binding and dimerization domains are located within
the COOH terminal two-thirds of the USF2 protein and are contained
within our truncated rUSF2 construct. As shown in Fig.
5 (open bars), when A549 cells were
cotransfected with the intact rSP-A-991:hGH
fusion gene and with pCMV-USF2, only a modest induction of hGH
expression was found compared with that observed with cotransfection of
the rSP-A
991:hGH fusion gene with "empty"
expression vector plasmid pCMV as control. As we observed previously
(14), when A549 cells were cotransfected with
rSP-A
991:hGH and with pCMV-USF1a, an
approximately fourfold induction of hGH expression was found.
Interestingly, when A549 cells were cotransfected with
rSP-A-991:hGH and with pCMV-USF2 and pCMV-USF1a
in combination, a synergistic eightfold induction of rSP-A
promoter activity was observed. By contrast, when A549 cells were
cotransfected with rSP-A
976:hGH (lacking the
DBE; hatched bars), little or no induction of fusion gene expression
was found on cotransfection of rUSF2 and rUSF1 expression vectors,
alone or in combination. When the cells were transfected with
rSP-A-991PBE(
):hGH (containing a mutation in
the PBE; solid bars) fusion gene expression was essentially
undetectable and was unchanged on cotransfection of pCMV-USF2 or
pCMV-USF1a, alone or in combination. These findings suggest that
rUSF1/rUSF2 synergistically increase rSP-A promoter activity
by binding to the DBE and PBE. Furthermore, synergistic activation of
the rSP-A promoter is dependent on the integrity of both
E-boxes.
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DISCUSSION |
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The 5'-flanking region of the rabbit SP-A gene contains two structurally similar E-box-like sequences, termed DBE and PBE that compete for binding to rabbit lung nuclear proteins (13). Deletion or mutation of the DBE or PBE in rSP-A:hGH fusion genes reduced basal and cAMP induction of rSP-A promoter activity in transfected type II cells in primary culture. The DBE contains the E-box core sequence CACGTG, which is known to interact with a subset of gene regulatory proteins containing a bHLH structure. The PBE contains the sequence CTCGTG, which differs by one nucleotide from the DBE (13). Because the PBE and the DBE compete for lung nuclear protein binding (13), we considered it likely that similar or identical proteins bind to these elements.
A fetal rabbit lung cDNA expression library was screened using the radiolabeled PBE as probe; two cDNA inserts were isolated that encode proteins that specifically bind to the DBE and PBE elements. We previously reported (14) that one of these cDNAs encodes the rabbit homolog of hUSF1 (17, 30, 31). In the present study, we found that the second cDNA encodes the rabbit homolog of USF2/Fos-interacting protein (6, 34, 36). USF1 (43 kDa) and USF2 (44 kDa) are heat-stable, ubiquitously expressed proteins that bind as a dimer to the core E-box motif CACGTG (30, 31). Previously, we observed binding to the DBE and PBE of heat-stable type II cell nuclear proteins of molecular mass of 69,000, 45,000 and 22,000; the 45-kDa protein(s) appeared to be the predominant species and to bind as a dimer (13). In EMSA of rabbit type II cell nuclear protein binding to the PBE and DBE, we found that antibodies raised against rUSF1 supershifted a major portion of the binding complexes (14). In the present study, we observed a similar phenomenon using antibodies that are specific for rUSF2. These findings, together with those from yeast two-hybrid studies, indicating preferential interaction of USF1/USF2 heterodimers compared with homodimers, suggest that USF1/USF2 heterodimers comprise a principal component of the complex of type II cell nuclear proteins that bind to the DBE and PBE.
The results of lung cell transfection studies also provide evidence for the functional importance of binding of USF1/USF2 heterodimers to the DBE and PBE in activation of the rSP-A promoter. Cotransfection of A549 cells with pCMV:USF2 only modestly increased SP-A-991:hGH expression over basal levels. Whereas cotransfection of pCMVUSF1a caused an approximately fourfold induction of reporter gene expression, cotransfection of both USF1a- and USF2-containing expression vectors caused an approximately eightfold increase in rSP-A promoter activity. These findings correlate with those obtained using the yeast two-hybrid system, in which the efficiency of USF1 and USF2 heterodimerization was approximately twice that for USF1 homodimerization and approximately fourfold greater than that for USF2 homodimerization. It should also be noted that the transactivation domains in USF1 have been reported to be relatively weak (12); therefore, heterodimerization of USF1 and USF2 may be critical in the regulation of SP-A gene expression. Our findings are in accord with those of previous studies suggesting that USF1/USF2 heterodimers comprise the predominant DNA-binding form in a variety of cells and tissues, and that USF1 and USF2 homodimers constitute only a minor component of the USF binding activity (33, 36).
A critical functional role of USF1/USF2 heterodimers is also suggested
from studies of mice carrying targeted deletions in the Usf1
and Usf2 genes (33). Whereas,
Usf1/
mice were viable and fertile,
Usf2
/
mice manifested a pronounced growth
defect. Interestingly, USF2 expression was increased in USF1-null mice,
whereas USF1 expression was markedly decreased in USF2-null mice. This
suggests USF1 negatively regulates USF2 expression, whereas USF2
positively regulates USF1 expression and that the apparently normal
growth in the Usf1
/
mice may be due to a
compensatory upregulation of USF2 (33). In contrast to the
single knockouts, an embryonic lethal phenotype was observed in mice
that were homozygous for the Usf2 mutation and either
homozygous or heterozygous for the mutation in Usf1 (33). There was no indication in that report as to the
stage of development that embryonic demise occurred.
SP-A gene transcription is developmentally regulated in fetal rabbit lung and is first detectable in lung nuclei on day 24 of gestation (term = day 31), reaching maximal levels by day 28 (7). By use of quantitative RT-PCR, we previously observed that rUSF1 mRNA is readily detectable on day 21 of gestation, reaches peak levels on day 23, just before the time of activation of rSP-A gene transcription, and subsequently declines to relatively low levels on day 28 (14). By contrast, in the present study, we found that rUSF2 mRNA levels are relatively low in fetal rabbit lung on day 21 of gestation, increase approximately twofold on day 25 and reach maximal levels on day 28, in concert with the time of maximal induction of rSP-A gene expression. These findings suggest that the temporal induction of SP-A gene transcription in fetal rabbit lung may be regulated, in part, by the relatively high levels of USF1 and USF2 on day 24 of gestation, facilitating their heterodimerization and binding to enhancers in the 5'-flanking region of the rSP-A gene. Because the findings in gene-targeted mice suggest that USF1 negatively regulates Usf2 gene expression (33), it is possible that the developmental increase in rUsf2 expression in rabbit fetal lung occurs only after the levels of rUSF1 begin to decline.
In studies using transgenic mice carrying rSP-A:hGH fusion
genes containing various amounts of 5'-flanking DNA from the rabbit SP-A gene, our laboratory has found that fusion genes
containing as little as 378 bp of rSP-A 5'-flanking DNA
are expressed in a lung-specific manner, specifically in type II cells
and in bronchoalveolar epithelial cells (2). The
rSP-A
378:hGH transgenes are also
developmentally regulated in concert with the endogenous mouse
SP-A gene (2). These findings indicate that
response elements within the 378 bp 5'-flanking region are most
critical for tissue/cell-specific and developmental regulation of
rSP-A expression and suggest that the PBE may play a more
crucial role than the DBE in this regulation. Previously, we observed
that a fusion gene containing both the DBE and PBE
(rSP-A
991:hGH) was induced ~25-fold by cAMP
treatment of transfected type II cells. Whereas mutation of the PBE
within this construct markedly reduced basal and essentially eliminated
cAMP induction of rSP-A promoter activity, removal of the
DBE reduced both basal and cAMP-induced expression to levels similar to
that observed using an rSP-A
378:hGH fusion
gene so that a 15-fold stimulation by cAMP of rSP-A promoter activity was still observed (13). We therefore suggest
that the DBE serves as an enhancer required for high levels of basal and cAMP stimulated SP-A expression in cultured type II
cells, whereas the PBE, together with other response elements within the
378-bp 5'-flanking sequence, plays a more essential role. It
should be noted that we have identified another E-box sequence (median
binding element; MBE) (CACGTG) at
340 bp within the rSP-A 5'-flanking region that we have found to also be important for cAMP
regulation of rSP-A promoter activity in transfected type II
cells (E. Gao and C. R. Mendelson, unpublished
observations). It is possible that within chromatin, USF1/USF2
proteins bound to the PBE act cooperatively with those bound to the MBE
and to other critical response elements to mediate appropriate temporal and spatial regulation of rSP-A gene expression. Although
USF1 and USF2 are ubiquitously expressed proteins, their patterns of developmental regulation and the enhanced levels of USF1/USF2 DNA-binding activity in type II cells, compared with lung fibroblasts, may serve an important role in developmental and cell-specific expression of the SP-A gene in fetal lung.
USF2 and USF1 contain two protein interacting domains, HLH and LZ. It should be noted that USF2 was initially isolated by its interaction with the LZ domain of c-Fos, and was found to cooperatively interact with c-Fos in activation of an activator protein-1-responsive promoter (6). As mentioned previously, we have observed that cAMP stimulation of SP-A promoter activity in transfected type II cells is critically dependent on the integrity of a number of response elements, including a putative nuclear receptor binding site (CRESP-A) (28, 38), a TTF-1-binding element (21), the PBE (13), and a GT-box that binds Sp1; mutagenesis of any one of these sites severely reduces basal and cAMP stimulation of SP-A promoter activity (39). Cooperative interaction of USF2/USF1 with other transcription factors bound to the proximal SP-A promoter is, therefore, likely to play a critical role in developmental, hormonal, and tissue-specific regulation of SP-A gene expression.
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ACKNOWLEDGEMENTS |
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The authors gratefully acknowledge the expert assistance of Margaret Smith in the isolation and culture of lung type II cells and fibroblasts.
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FOOTNOTES |
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This research was supported by National Heart, Lung, and Blood Institute Grant R37 HL-50022 (to C. R. Mendelson) and by Grant-in-Aid 94R-084 from the American Heart Association, Texas Affiliate (to E. Gao).
Present address for J. L. Alcorn: Dept. of Pediatrics, Univ. of Texas Medical School at Houston, 6431 Fannin, Ste. 3-222, Houston, TX 77030.
Address for reprint requests and other correspondence: C. R. Mendelson, The University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd., Dallas, TX 75390-9038 (E-mail: cmende{at}biochem.swmed.edu).
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. Section 1734 solely to indicate this fact.
First published February 7, 2003;10.1152/ajplung.00219.2002
Received 9 July 2002; accepted in final form 21 January 2003.
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