Division of Pulmonary Biology, Children's Hospital Medical Center, Cincinnati, Ohio 45229
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ABSTRACT |
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The regulatory role
of activator protein-1 (AP-1) family members in mouse surfactant
protein (SP) B (mSP-B) promoter function was assessed in the mouse lung
epithelial cell line MLE-15. Expression of recombinant Jun B and c-Jun
inhibited mSP-B promoter activity by 50-75%. Although c-Fos
expression did not alter mSP-B transcription, Jun D enhanced mSP-B
promoter activity and reversed inhibition of mSP-B by c-Jun or Jun B. A
proximal AP-1 binding site (18 to
10 bp) was identified
that overlaps a thyroid transcription factor-1 binding site. Mutation
of this proximal AP-1 site blocked both Jun B inhibition and Jun D
enhancement and partially blocked c-Jun inhibition of promoter
activity. Promoter deletion mutants were used to identify additional
sequences mediating the inhibitory effects of c-Jun in the distal
region from
397 to
253 bp. The AP-1 element in this
distal site (
370 to
364 bp) is part of a composite
binding site wherein AP-1, cAMP response element binding protein,
thyroid transcription factor-1, and nuclear factor I interact. Point
mutation of the distal AP-1 binding site partially blocked
c-Jun-mediated inhibition of the SP-B promoter. Both stimulatory (Jun
D) and inhibitory (c-Jun/Jun B) effects of AP-1 family members on mSP-B
promoter activity are mediated by distinct
cis-acting elements in the mSP-B
5'-flanking region.
surfactant protein B; activator protein-1; pulmonary surfactant; gene transcription; respiratory epithelium; regulatory promoter
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INTRODUCTION |
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SURFACTANT PROTEIN (SP) B is a 79-amino acid amphipathic polypeptide associated with surfactant phospholipids in the alveolus (for a review, see Ref. 41). SP-B enhances the rate of spreading and stability of phospholipids crucial to reducing alveolar surface tension. Both human and mouse SP-B (mSP-B) mRNAs are expressed in nonciliated bronchiolar and alveolar type II epithelial cells (9, 27). SP-B is critical for postnatal pulmonary adaptation. Transgenic mice homozygous for a null mutation in the SP-B gene failed to expand their lungs at birth (8). Likewise, human infants with mutations in the SP-B gene succumb to respiratory failure in the neonatal period (23). Lung compliance was decreased in heterozygous SP-B gene-targeted mice (7) associated with a 50% reduction in SP-B mRNA and protein, suggesting that reduction in SP-B alters pulmonary function. SP-B concentrations are reduced in various clinical conditions including respiratory distress syndrome in infants and acute respiratory distress syndrome in adults (12, 30). The temporal, spatial, and humoral regulation of SP-B synthesis are controlled at both transcriptional and posttranscriptional levels (41).
The expression of activator protein-1 (AP-1) family members is
activated by phorbol esters (19). The potential role of AP-1 in the
regulation of the SP-B gene is supported by the finding that phorbol
ester inhibited SP-B transcription (31). SP-B mRNA and SP-B protein
synthesis were inhibited by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) and by tumor
necrosis factor- in H441-4 cells and mouse lung
(29, 31).
A variety of other growth factors, hormones, mitogens, and cytokines are also known to induce expression or activate members of the AP-1 family. In the lung, c-Jun mRNA is expressed at higher basal levels than in other tissues (33, 34). Agents that produce reactive oxygen radicals such as H2O2 or asbestos are strong inducers of c-Jun in lung cells (15, 39). Oxygen radicals cause injury to the lung epithelium (38) and increase SP-B expression in bronchiolar epithelial cells but decrease SP-B expression in alveolar epithelium (43).
A proximal AP-1 binding sequence has been identified in the human SP-B promoter (4, 22, 28). AP-1 binding sites have been found in the promoters of other lung-specific genes including Clara cell secretory protein (35) and SP-D (32). The present study identified two distinct regions of the mSP-B promoter bearing AP-1 binding sites that bind and function through different AP-1 family members.
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MATERIALS AND METHODS |
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Plasmid construction and mutagenesis.
The mSP-B promoter constructs were cloned in the pBLCAT6 reporter
vector. This promoter template, p1797/+42/CAT, and the primer
bearing the appropriate mutation were used for site-directed
mutagenesis as described by Kunkel (18). The sequence of the proximal
AP-1 site was 5'-GAG CCC
A
GTA GGG TAC-3' (
25 to
10 bp), and the proximal
mutant TG-AP-1 primer sequence was 5'-GAG CCC
AA GTA GGG TAC-3'
(lowercase letters indicate bases that introduced mutation). The AP-1
mutant in the distal promoter region (p
653/AP-1/CAT) was
generated by PCR site-directed mutagenesis as previously described (5),
with the p
653/+42/CAT mSP-B promoter construct as a template.
Primers bearing 5' Hind III or
3' Sal I sites were made to the
653 and +42 regions of the promoter, respectively. The wild-type
(WT) sequence of the distal AP-1 binding site was 5'-CTT ACC
A GAG CCA GGA-3', and the
sequence of the primer containing a mutation in that site was
5'-CTT ACC
A GAG CCA GGA-3'. All mutants were verified by sequencing.
A series of mSP-B promoter constructs containing 5' deletions
(p1173, p
753, p
653, p
543, p
415,
p
397, p
353, and p
297 SP-B/CAT) were generated with
the WT p
1797/+42/CAT plasmid template and PCR linker primers to
the corresponding regions of the promoter bearing 5'
Hind III or 3'
Sal I restriction sites. PCR products were gel purified and cloned in the pBLCAT6 reporter vector. Correct mutants were verified by sequencing.
Cell culture, transfections, and reporter gene
assay. The mouse clonal cell line MLE-15 was derived
from lung tumors produced in transgenic mice expressing SV40 large T
antigen under the control of an SP-C lung-specific promoter (42). These
cells were propagated in HITES (hydrocortisone, insulin, transferrin,
estradiol, and sodium selenite) medium containing 4%
fetal bovine serum (FBS; Sigma) as previously described (42). Transient
transfections were done with the calcium phosphate precipitation method
as previously described (3) in either 100-mm plates in duplicate or
six-well Falcon plates in triplicate samples. The SP-B
promoter/chloramphenicol acetyltransferase (CAT) reporter constructs
(2.3 µg/well or 12 µg/plate) were cotransfected with pCMV--gal
(1.25 µg/well or 7 µg/plate) as an internal transfection control.
The cotransfection experiments were performed with 0.83 µg/well or
2.5 µg/plate of each or a combination of pRSV/c-Jun, pRSV/Jun B,
pRSV/Jun D, and pSVE/c-Fos expression plasmids (kind gifts from Dr. M. Yaniv, Unité des Virus Oncogènes, Institut Pasteur, Paris,
France) or empty pRSV plasmid as indicated. To correct for variation in
transfection efficiency, the same amount of -gal units was used in
CAT enzyme analysis. Cell extracts were prepared with three freeze-thaw
cycles in 50-100 µl of 0.25 M Tris (pH 7.8)/well. CAT assays
were performed as previously described (11). Thin-layer chromatograms
of
[14C]chloramphenicol
and its acetylated derivatives were quantitated with a Molecular
Dynamics phosphorimager (Storm 680). CAT activity was calculated as a
percentage of either the WT promoter or the promoterless CAT control
(pBLCAT6) activity as indicated. CAT activity is expressed as mean ± SE from experiments performed in triplicate or several
experiments performed in duplicate.
Preparation of nuclear extracts. Nuclear extracts for electrophoretic mobility shift assays (EMSAs) were prepared with the high-salt procedure of Schreiber et al. (36), with modifications. Briefly, confluent monolayers of MLE-15 cells were scraped off on ice, washed in ice-cold PBS, and pelleted at 3,000 rpm for 5 min. The cell pellets were washed in PBS and lysed in two volumes of buffer A [10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 1.5 mM MgCl2, 0.2% Nonidet P-40, 1 mM dithiothreitol (DTT), and 0.5 mM phenylmethylsulfonyl fluoride (PMSF)] by gentle vortexing. Nuclei were isolated by centrifugation at 3,000 rpm for 5 min. Nuclear pellets were then resuspended, and the protein was extracted in one volume of high-salt buffer B (20 mM HEPES, pH 7.9, 420 mM NaCl, 0.1 mM EDTA, 1.5 mM MgCl2, 25% glycerol, 1 mM DTT, and 0.5 mM PMSF). Nuclear proteins were then recovered by centrifugation at 14,000 rpm for 10 min. Protein concentration was determined by the bicinchoninic acid method (Sigma) with BSA as a standard. Protein concentration in the nuclear extracts was typically 5-10 µg/µl.
EMSA. The gel shift probes were
generated by annealing synthetic oligonucleotides (typically 24 mers)
at a concentration of 10 µM in buffer
C (10 mM Tris · HCl, pH 7.5, 10 mM
MgCl2, and 50 mM NaCl).
Double-stranded products were gel purified with 4% Biogel and Mermaid
kit (BIO 101). The concentration of annealed oligonucleotides was then
adjusted to 2 pmol/µl, and 1 µl was end labeled with
[-32P]ATP and T4
polynucleotide kinase. The sequences of competitors used in the EMSAs
were AP-1 collagenase (1, 21), 5'-CGC TTG ATG AGT CAG CCG
GAA-3'; AP-1 osteocalcin (37), 5'-TCG ACA CCC GGT GAG TCA
CCT AGA-3'; thyroid transcription factor-1 (TTF-1) thyroglobulin
(6), 5'-CAC TGC CCA GTC AAG TGT TCT TGA-3'; and cAMP
response element binding protein (CREB; Promega), 5'-AGA GAT TGC
CTG ACG TCA GAG AGC TAG-3'. The probes were purified with Nick
G-50 columns (Pharmacia Biotech, Uppsala, Sweden) and diluted to 20,000 dpm/µl. Nuclear extracts (5 µg) were preincubated in the presence
and absence of unlabeled competitors (100-fold excess or as indicated)
in binding buffer [12 mM HEPES (pH 7.9), 4 mM Tris · HCl (pH 7.9), 25 mM KCl, 5 mM
MgCl2, 1 mM EDTA, 12% glycerol, 4 mM DTT, 40 ng/µl of poly(dI-dC), and 0.2 mM PMSF] for 15 min at
room temperature. Four microliters of probes were added, and the
mixture was incubated for an additional 20 min. The reactions with
recombinant proteins were performed in the absence of nuclear extract.
Bacterially expressed TTF-1 homeodomain was a kind gift from Dr. R. DiLauro (Stazione Zoologica Anton Dohrn, Villa Comunale, Naples,
Italy). Recombinant CREB DNA binding domain was purchased from Santa
Cruz Biotechnology. All antibodies to the AP-1 family members (c-Jun, Jun B, Jun D, and c-Fos) were purchased from Santa Cruz
Biotechnology and typically added before the probe and incubated overnight at 4°C. Protein-bound and free probes were resolved with
5% nondenaturing gel electrophoresis. The gels were dried and exposed
on X-ray film (Kodak) for 5 h at room temperature.
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RESULTS |
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Proximal region of the mSP-B promoter contains
overlapping AP-1 and TTF-1 binding sites. Conserved
consensus binding sites for the AP-1 family of transcription factors
are located in close proximity to the respective TATA boxes in both the
mSP-B and human SP-B promoters. Previous in vitro DNase I footprinting
analysis with H441 lung adenocarcinoma and HeLa cell nuclear extracts
identified a protected region (+15 to +33) over a potential AP-1
binding site in the human SP-B promoter (4). Close inspection of the murine promoter sequence revealed the presence of potential overlapping binding sites for AP-1 (TGACTCA) and TTF-1 (CAAG) located at 18 to
10 bp from the transcription start site (Fig.
1A).
Incubation of an oligonucleotide probe corresponding to this region
with MLE-15 nuclear extracts revealed two DNA-protein complexes of similar mobility (Fig. 1B,
lane 1). As shown in Fig.
1B, formation of the slower mobility
complex was inhibited by 100-fold molar excess of a cold
oligonucleotide competitor bearing the AP-1 (lane 3) consensus sequence from the collagenase gene
promoter (1, 21). Formation of the faster mobility complex was
inhibited by 100-fold molar excess of an oligonucleotide competitor
containing the TTF-1 binding site (Fig.
1B, lane
4) from the thyroglobulin gene promoter (6). This
element (
18 to
10 bp) also bound recombinant TTF-1
homeodomain protein. Therefore, both AP-1 and TTF-1 factors bind in
close proximity to each other at the proximal (
18 to
10
bp) element in the murine SP-B promoter.
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To further characterize the binding of AP-1 and TTF-1 to this region,
we generated oligonucleotides containing point mutations to abolish the
binding of the AP-1 complex. As shown in Fig.
1B, formation of the slower-mobility
AP-1 complex was abolished when the oligonucleotide TG-AP-1 bearing a
point mutation at the AP-1 site (TGACTCA gtACTCA) was used as
a probe for EMSA analysis (lanes
6-10), leaving
the faster-mobility TTF-1 complex intact. Weak competition was observed
when an AP-1 cold competitor from the collagenase promoter was used
(Fig. 1B, lane
8). This is probably due to a cryptic TTF-1 site in
the AP-1 competitor (CTTG). Antibodies to the AP-1 family members did
not supershift the TG-AP-1 probe, confirming that the point mutation
abolished the ability to interact with the AP-1 site (data not shown).
The TTF-1 cold competitor partially inhibited the complex formed with
the TG-AP-1 probe and MLE-15 cell nuclear proteins (Fig.
1B, lane
9). The TG-AP-1 mutant retained the ability to bind
the recombinant TTF-1 homeodomain (Fig.
1B, lane
10). Similarly, binding of TTF-1 was blocked by a
mutation in the TTF-1 consensus site (G-TTF-1 mutant) that left the
AP-1 site intact (Sever-Chroneos, Bachurski, and Whitsett, unpublished
observations). These findings suggest that AP-1 and TTF-1
proteins can bind to the
18- to
10-bp site independently of one another.
Preliminary studies with S1 nuclease protection analysis showed that MLE-15 cells expressed mRNAs of AP-1 family members c-Jun, Jun B, Jun D, c-Fos, and Fos B (data not shown). Supershift assay and transient transfections with expression constructs were used to investigate the role of different AP-1 family members in the regulation of the murine SP-B gene in MLE-15 cells. Antibodies to Jun B and Jun D supershifted the WT probe containing the proximal AP-1 site sequence (Fig. 1C, lanes 4 and 5). Antibodies to the c-Jun and c-Fos DNA binding domains partially inhibited the protein-DNA complex (Fig. 1C, lanes 3 and 6, respectively), and an antibody to the NH2 terminus of c-Jun produced a weak supershift (data not shown). Thus the complex binding to the proximal AP-1 site in MLE-15 nuclear extracts consists primarily of Jun B, Jun D, and possibly c-Jun and c-Fos in vitro.
Distinct regulation of the mSP-B promoter by different
AP-1 family members. To further investigate the
significance of AP-1 family members binding to the proximal AP-1 site,
expression vectors for c-Jun, Jun B, Jun D, and c-Fos were
cotransfected with the p1797/+42/CAT construct in MLE-15 cells.
Coexpression of c-Jun decreased mSP-B promoter activity 70-80%
(Fig.
2A).
Jun B expression inhibited transcription to a lesser extent (~50%).
Jun D enhanced mSP-B promoter activity (~125%), whereas c-Fos did
not alter mSP-B promoter activity (Fig.
2A).
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To determine whether specific AP-1 family members act through the
proximal AP-1 binding site, a mutant mSP-B promoter construct, p1797/TG-AP-1/CAT, was cotransfected with AP-1 expression
vectors in MLE-15 cells. As shown in Fig.
2B, inhibition of the mSP-B promoter
by Jun B was blocked by mutation of the proximal AP-1 binding site.
Activation of mSP-B promoter activity by Jun D was also blocked by
mutation of the proximal AP-1 site. This indicates that Jun B and Jun D
AP-1 family members interact with the proximal AP-1 site in MLE-15
cells. Mutation of the proximal AP-1 site partially blocked
c-Jun-mediated inhibition of mSP-B promoter activity (Fig.
2B), indicating that c-Jun may act
through additional AP-1 sites in the mSP-B promoter.
The AP-1 family of transcription factors binds to DNA sites as
homodimers or heterodimers (2). Therefore, distinct combinations of
AP-1 family members were coexpressed with mSP-B p1797/+42/CAT to
determine whether their coexpression alters mSP-B gene transcription. Coexpression of both c-Jun and Jun B constructs inhibited mSP-B promoter activity by 75%. Coexpression of c-Fos did not alter the
inhibitory effect of c-Jun or Jun B. However, coexpression of Jun D
partially blocked the inhibitory effects of both c-Jun and Jun B (Fig.
3). Therefore, AP-1 family
members have distinct effects on the activity of the mSP-B promoter.
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Specificity and localization of the mSP-B promoter
region mediating inhibitory effects of c-Jun. Transient
transfection of the pRSV/c-Jun expression vector in MLE-15 cells
inhibited SP-B promoter function in vitro. Increasing concentrations of
the pRSV/c-Jun expression construct were cotransfected with the mSP-B
WT (p1797/+42/CAT) promoter. SP-B promoter activity was
inhibited in a dose-dependent manner with cotransfection of
0.2-0.5 µg of c-Jun expression vector (Fig.
4A). The
inhibition of promoter activity by c-Jun reached a threshold (~75%)
with 1.0 µg of cotransfected c-Jun. Cotransfection of 2.5 and 5 µg
of pRSV/c-Jun did not further inhibit mSP-B promoter activity. This
indicates that c-Jun is a specific inhibitor of basal mSP-B promoter
activity in MLE-15 cells.
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Transient cotransfection of p1797/TG-AP-1/CAT and pRSV/c-Jun in
MLE-15 cells suggested the presence of a distinct promoter site
mediating inhibition of SP-B activity by c-Jun (Fig.
2B). To identify the site of c-Jun
inhibitory effects, a series of five deletions of the mSP-B promoter
were tested in transient transfection assays in MLE-15 cells in the
presence and absence of pRSV/ c-Jun. As shown in Fig.
4B, most of the inhibitory effects of
c-Jun were lost after deletion of a region located between
397
and
353 bp from the start of transcription, indicating a potential interaction of c-Jun with this region. Interestingly, basal
promoter activity was lost with the deletion of promoter sequences from
415 to
353 bp. This region was also protected by nuclear
proteins from MLE-15 cells in in vitro DNase I footprinting assays
(Sever-Chroneos, Bachurski, and Whitsett, unpublished observations).
Analysis of distal 370 to
364 bp AP-1
binding site. The mSP-B promoter in the region from
397 to
353 bp contains potential binding sites for a
number of transcription factors that influence SP-B promoter activity.
A potential AP-1 binding site overlaps with CREB, and both are closely
apposed to a TTF-1 binding consensus (
365 to
362 bp) and
a half binding site (
360 to
357 bp) for the nuclear
factor I (NF-I) family members (Fig.
5A).
Disruption of TTF-1 and NF-I binding to these sites abolished basal
mSP-B promoter activity (unpublished observations). The interaction of
TTF-1 and NF-I transcription factors with the promoter region from
397 to
353 bp is likely to be responsible for the loss of
basal promoter activity with the deletion of promoter sequences from
397 to
353 bp (Fig.
4B).
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To investigate binding of AP-1 factors to this region, we used a WT
oligonucleotide probe to the 377- to
353-bp region in the
EMSA experiment. A single complex was detected with this probe in the
presence of MLE-15 nuclear proteins (Fig.
5B, lanes
1 and 2). In
addition, an oligonucleotide bearing a point mutation (CTGCGTCA
CaaCGTCA) in the distal AP-1 site (
370 to
364
bp) also formed a single complex with MLE-15 nuclear proteins (Fig.
5B, lanes 3 and 4). The
formation of this complex was not inhibited by the presence of the AP-1
consensus from an osteocalcin promoter, but competition with a CREB
consensus oligonucleotide partially inhibited complex formation and
recombinant CREB formed a complex with an AP-1 mutant probe (Fig.
5B, lanes
5-7),
suggesting that CREB interacts with the
377 to
353 bp
binding site independently from AP-1. Moreover, the complex formed by
the AP-1 mutant probe was inhibited by both TTF-1 and NF-I unlabeled
competitors (Sever-Chroneos, Bachurski, and Whitsett, unpublished
observations). A point mutation in the putative TTF-1 and NF-I binding
sites (TTF-1/NF-I mut) formed a complex that interacted with antibodies
to both Jun D and c-Jun (Fig. 5B,
lanes
8-11).
Mutation of the distal AP-1 binding site (p653/AP-1/CAT)
increased basal promoter activity fivefold in MLE-15 cells (Fig. 6). This indicates that the distal AP-1
(
370 to
364 bp) site is an inhibitory promoter element.
To test whether c-Jun inhibition is mediated through the distal AP-1
binding site, the WT p
653/+42/CAT and distal mutant
p
653/AP-1/CAT mSP-B promoter constructs were transfected with
and without the pRSV/c-Jun expression vector (Fig. 6). Transient
expression of c-Jun in MLE-15 cells inhibited WT promoter activity by
80% (Fig. 6). Similar to the cotransfection of the proximal AP-1 site
mutant promoter, CAT activity of the distal AP-1 mutant promoter was
inhibited by 50% with c-Jun, indicating that the distal AP-1 element
(
370 to
364 bp) is involved in, but is not sufficient
for, c-Jun-mediated inhibition of promoter activity. Interestingly,
coexpression of Jun D with c-Jun overcame the c-Jun-dependent
inhibition of p
653/AP-1/CAT (Fig. 6) as seen with the WT
promoter (Fig. 1B). Mutation of the
distal AP-1 site did not alter interaction of Jun D with the mSP-B
promoter, suggesting that Jun D is able to counteract the remaining
c-Jun inhibition through the interaction with the proximal AP-1 binding
site.
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In summary, AP-1 family members interact with both proximal (18
to
10 bp) and distal (
370 to
364 bp) mSP-B
promoter sites. mSP-B promoter activity is inhibited by the interaction
of Jun B with the proximal AP-1 binding site and by the interaction of c-Jun with both the proximal and distal AP-1 binding sites.
Cotransfection with Jun D stimulates SP-B promoter function and
antagonizes the inhibitory effects of c-Jun and Jun B on mSP-B promoter activity.
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DISCUSSION |
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Two distinct sites of AP-1 family member interaction with the mSP-B
promoter were identified. A proximal AP-1 binding site that overlaps
with a TTF-1 binding site is located from 18 to
10 bp
from the transcription start site (Fig.
1A). Similarly, an AP-1 consensus
is situated at +20 to +26 bp in the human SP-B promoter (4),
demonstrating the conservation of this motif in the proximal region of
human SP-B and mSP-B promoters. Inhibition of mSP-B promoter activity
by Jun B was mediated by the proximal (
18 to
10 bp) AP-1
site, whereas the inhibitory effect of c-Jun was mediated by both the
proximal and distal regions from
397 to
253 bp in the
mSP-B promoter. A cis-acting element
located at
370 to
364 bp in the distal promoter region
contained superimposed AP-1 binding/CREB sequences overlapping with
binding sites for other transcription factors. Jun D interacted with
the proximal (
18 to
10 bp) AP-1 binding site to enhance
promoter activity and block the inhibitory effects of both c-Jun and
Jun B. These results suggest a complex interaction of distinct AP-1
family members binding to at least two regions of the mSP-B promoter.
In this work, we show that c-Jun inhibits mSP-B promoter activity in a
dose-dependent manner by interacting with both the proximal (18
to
10 bp) and distal (
397 to
253 bp) regions of
the SP-B promoter. Moreover, the proximal AP-1 binding site mediated
inhibition of the mSP-B promoter by Jun B. Jun D enhanced promoter
activity through the proximal AP-1 site in vitro. In another system,
different AP-1 family members also have distinct effects on
estrogen-dependent transcription. In that system, the expression of
c-Jun, Jun B, and c-Fos inhibited estrogen-induced estrogen receptor
activity, whereas Jun D did not affect promoter activity (10).
Deletion mutants of the mSP-B promoter cotransfected with c-Jun
expression vector pinpointed an additional site of c-Jun inhibitory effects between 397 and
253 bp. However, with the use of
MLE-15 cell extracts, the binding of endogenous c-Jun to the proximal and distal promoter elements was weak. Consistent with this
observation, MLE-15 cells expressed lower levels of c-Jun mRNA compared
with Jun B and Jun D mRNAs (Sever and Whitsett, unpublished
observations). In addition, the level of Jun D mRNA in the mouse lung
was previously estimated to be 5-10 times higher than the level of
c-Jun mRNA (13). Therefore, the high levels of endogenous SP-B
expression in MLE-15 cells (42) correlate with the low levels of c-Jun and increased Jun D expression in these cells.
Sequence analysis of the 370- to
364-bp promoter element
identified an imperfect AP-1 and a half CREB site. A previous study (44) suggested that CREB inhibits human SP-B promoter activity. Another
potential CREB site is conserved at
78 bp of human SP-B (28) and
at
80 bp of the murine promoter. Interestingly, recombinant CREB
DNA binding domain also binds to the WT proximal AP-1 mSP-B oligonucleotide probe from
25 to
2 bp (data not shown).
Similar findings were reported wherein CREB sites mediated c-Jun
inhibition of the placental hormone chorionic gonadotropin
and
gene transcription (26) and c-Jun and c-Fos inhibition of Myo D
transcription (25). Jun D activates the proenkephalin promoter through
the CREB site, and that activation is dependent on protein kinase A
activation but is blocked by cotransfected Jun B (16).
The expression of Jun D alone in MLE-15 cells enhanced SP-B promoter activity, and Jun D antagonized the c-Jun- and Jun B-mediated inhibition of SP-B promoter activity. Cotransfection of Jun D with c-Jun restored promoter activity to 75% of the control value. Cotransfection of Jun D with Jun B abolished the inhibitory effect of Jun B on SP-B promoter activity. This observation is likely explained if, for example, heterodimers of c-Jun and Jun B with c-Fos are active inhibitors of SP-B gene transcription. On the other hand, heterodimers of Jun D with either c-Jun or Jun B are likely inactive as repressors of SP-B promoter activity. Similarly, in liver cells (14), heterodimerization of Jun B with c-Fos activate, whereas Jun B and liver regenerating factor-1 dimers repress the c-Fos/ c-Jun-mediated activation. We speculate that expression of distinct combinations of AP-1/Jun and CREB family members in the alveolar and bronchiolar epithelia may have distinct effects on mSP-B gene regulation.
In the mSP-B promoter, AP-1/CREB and TTF-1 share overlapping binding
sites located from 18 to
10 and
370 to
364
bp and are likely to compete for binding to their respective sites. It is well established that repression of transcription may occur by the
prevention of binding (24). The repression is indirect when a repressor
binds to the activator binding site and thus prevents the activator
from binding (for a review, see Ref. 20). For example, c-Jun and c-Fos
inhibit osteocalcin gene transcription by preventing retinoic
acid-receptor binding to the same sequence (37). It is conceivable that
the binding of the negative regulators c-Jun, Jun B, and CREB may
compete for binding with TTF-1 or NF-I activators. We speculate that
binding of distinct AP-1 heterodimers to the proximal and distal AP-1
sites of the mSP-B promoter may either allow or interfere with binding
of other transcriptional activators such as TTF-1 to overlapping sites.
The repression of mSP-B gene transcription may thus be regulated by a variation in the concentration or activity of factors such as c-Jun, Jun B, CREB, and Jun D. Because Jun D is highly expressed in the lung (13), formation of AP-1 heterodimers with Jun D may allow other transcriptional activators such as TTF-1 to bind to the overlapping site(s), enhancing SP-B gene expression. The dynamic equilibrium between the relative abundance of AP-1/CREB and TTF-1 factors therefore may provide a mechanism by which SP-B gene regulation may be modulated during cell differentiation, proliferation, or repair.
Recently, TPA has been shown to regulate human SP-B expression by
cytoplasmic trapping of TTF-1 and hepatocyte nuclear factor-3 in A549
and H441 cells, respectively (17). Inhibitory effects of TPA on human
SP-B transcription were localized to the proximal promoter in H441
cells (40). This is consistent with a region containing TTF-1,
hepatocyte nuclear factor-3, and AP-1 binding sites in the human SP-B
promoter (4). Most commonly, AP-1 activity is associated with the
activation of the protein kinase C signal transduction pathway.
However, in other systems, AP-1 activity is also induced by a number of
cytokines (tumor necrosis factor-), growth factors (transforming
growth factor-
and fibroblast growth factor), and bacterial products
(lipopolysaccharide) (reviewed in Ref. 2), some of which are also known
inhibitors of SP-B expression (29). Moreover, in mouse models of oxygen
lung injury and adenoviral infection, SP-B mRNA expression is decreased
(43, 45). A modest reduction in SP-B in heterozygous SP-B knockout mice
alters lung function. Therefore, the elucidation of factors regulating
SP-B concentrations in the lung may have important clinical
implications (7). The AP-1 family members may interact with the SP-B
promoter to influence SP-B expression during development or after lung injury.
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ACKNOWLEDGEMENTS |
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We thank Dr. Moshe Yaniv for the activator protein-1 expression vectors, Dr. Roberto DiLauro for the recombinant thyroid transcription factor-1 homeodomain, and Dr. Robert Bohinski for initial input in this work.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute (NHLBI) Grant HL-60907 (to C. J. Bachurski); NHLBI Grant HL-38859; NHLBI Specialized Center of Research Grant HL-56387 (to J. A. Whitsett); and the American Lung Association (C. Yan).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: J. A. Whitsett, Children's Hospital Medical Center, Divisions of Neonatology and Pulmonary Biology, 3333 Burnet Ave., Cincinnati, OH 45229-3039 (E-mail: whitj0{at}chmcc.org).
Received 13 May 1998; accepted in final form 8 March 1999.
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