1 Institute for Environmental Medicine, University of Pennsylvania School of Medicine, Philadelphia 19104-6068; and Departments of 2 Pathology and 3 Molecular Pharmacology, MCP Hahnemann University School of Medicine, Philadelphia, Pennsylvania 19129
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ABSTRACT |
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Surfactant protein A (SP-A) is expressed in lung
alveolar type II cells and bronchiolar Clara cells. We have identified
two active regions in the promoter of the rat SP-A gene by deletion analysis of a plasmid containing 163 bp before the start of
transcription (163 bp), linked to a reporter gene. Constructs
were transfected into lung cell lines derived from each of the cell
types that produces SP-A. We found a novel region of promoter activity
at ~90 bp before the transcriptional start (SP-A
90).
Mutation of four nucleotides in SP-A
90 that are
highly conserved among species (
92 to
89 bp) decreased
expression of the SP-A construct by ~50% in both cell lines.
Electrophoretic mobility shift analysis showed specific binding to
SP-A
90 by nuclear proteins from the cell lines, as
well as from rat lung and liver. The electrophoretic mobility of the
bands shifted by lung nuclear proteins changed late in fetal
development. Although in the Clara cell line no reduction of promoter
activity was seen on deletion of the region upstream of
SP-A
90, in the type II cell line, deletion of
residues
163 to
133 did reduce activity by ~50%. This
region contains a recognition element for thyroid transcription factor-1 (TTF-1). Endogenous TTF-1 binding activity was substantially higher in the type II cell line than in the Clara cell line, but cotransfection of a TTF-1 expression plasmid enhanced expression of the
SP-A construct better in the Clara cell line than in the type II cell
line. These results suggest that the recognition element for TTF-1 has
varying activity in the lung cell lines of different origin due to the
availability of TTF-1.
gene regulation; transcription factors; lung development; NCI-H441 cells; MLE-15 cells
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INTRODUCTION |
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PULMONARY SURFACTANT lines the air-liquid interface of the smallest airways, alveoli, lowers surface tension, and prevents collapse on expiration (reviewed in Ref. 18). Surfactant is mostly lipid but also contains associated proteins (44). Surfactant protein A (SP-A) is produced in the lung in at least two cell types, alveolar type II epithelial cells and nonciliated bronchiolar Clara cells (15, 39). SP-A mRNA has also been observed in intestinal cells (34) and, more recently, in airway submucosal gland cells (35).
Several possible roles of SP-A in the regulation of pulmonary surfactant have been reported based on experiments in vitro (33, 40, 46). A recent report (21) that the SP-A gene can be knocked out in mice without any major effect on lung function, however, raises questions about its actual functions in vivo. SP-A and another surfactant protein, SP-D, are members of a family of molecules called collectins and may be involved in first-line immune defense (7). The SP-A knockout mice are, in fact, more susceptible than wild-type mice to bacterial pathogens (26, 27).
The mechanisms by which the expression of the SP-A gene is controlled are of interest. Surfactant (31) and SP-A (3) are produced only late in fetal lung development. This is true in all mammalian species examined (29). In humans, lack of surfactant is responsible for respiratory distress syndrome in premature infants (9). The mRNA encoding SP-A is not detectable by Northern analysis until embryonic day 18 of the 21-day gestation of the rat (10). Also, as mentioned above, SP-A expression is limited to certain cell types. The different cell types that express SP-A may regulate it differently. For example, it has been shown that exposure to hyperoxia increases SP-A mRNA in Clara cells but not in type II cells (13).
We have previously shown (37) that a short upstream region of the rat
SP-A gene (163 to +35 bp relative to the start of transcription)
confers promoter activity on a reporter gene in several cell lines.
Addition of regions further upstream in the SP-A gene diminished the
promoter activity.
In this paper, we describe the effects of various deletions and
mutations on the transcriptional activity of the rat SP-A promoter in
transient transfections of two lung cell lines: NCI-H441, thought to be
derived from a Clara cell (30, 36), and MLE-15, derived from a type II
cell (45). In general, regulation of transcriptional activity is
achieved through the association of trans-acting protein factors
(transcription factors) with particular DNA regulatory elements. We
have detected two regions in the rat SP-A promoter, the deletion or
mutation of which reduces transcription. One of these is a novel
element, SP-A90. The second region corresponds to a
domain located in the mouse SP-A gene that is bound and
trans activated by thyroid
transcription factor-1 (TTF-1; see Ref. 4). SP-A
90
is also bound by nuclear proteins. Changes in protein binding to
SP-A
90 were detected during late fetal and
neonatal rat lung development.
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METHODS |
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Deletion constructs. The starting
material for all deletion constructs of the promoter of the rat SP-A
gene was a plasmid, described previously (37), containing a fragment of
the gene extending from 1,013 bp upstream (1,013 bp) through 35 bp downstream (+35 bp) of the start of transcription. This fragment was
inserted 5' to the gene encoding chloramphenicol
acetyltransferase (CAT) at the Pst I
site of the commercial plasmid pCAT-Basic (Promega, Madison, WI). The
plasmid was linearized with Xho I,
which cut at
163 bp of the SP-A gene. The linearized plasmid DNA
was further digested at both ends with the bidirectional, double-strand
exonuclease Bal 31, slow (IBI, New Haven, CT) for times
varying from 15 to 120 min. The linearized plasmid was
next cut at an upstream Hind III site
within the multicloning region of the vector to remove the upstream
sequences of the SP-A gene (
1,013 bp through the end point of
Bal 31 digestion). After religation,
the plasmids containing the various deletions were grown in bacteria
and were analyzed by restriction digestion and sequencing (1).
Cell culture and transfection
analysis. The cell lines examined were NCI-H441, a
human adenocarcinoma thought to be derived from a bronchiolar Clara
cell (30, 36) and obtained from the American Type Culture Collection
(Manassas, VA), and MLE-15 derived from a mouse type II alveolar
epithelial cell (45), a gift from Dr. J. Whitsett, University of
Cincinnati (Cincinnati, OH). NCI-H441 cells were grown in RPMI 1640 medium with glutamine, supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% fetal calf serum. MLE-15 cells were
cultured in HITES (hydrocortisone, insulin, transferrin, estrogen, and
selenium) medium, modified as described (45). The cells were
transfected with SP-A-CAT plasmids at concentrations ranging from 1 to
10 µg/60-mm culture dish in different experiments, using Lipofectin
(GIBCO BRL, Grand Island, NY) in serum-free medium (Optimem; GIBCO BRL)
according to the supplier's instructions. One microgram of a plasmid
encoding Escherichia coli
-galactosidase driven by the cytomegalovirus (CMV) promoter (41), a
kind gift from Dr. Philip Ballard, Children's Hospital of Philadelphia
(Philadelphia, PA) was also added to each dish. In some experiments, we
added varying amounts of a TTF-1 expression plasmid that also utilized
the CMV promoter, CMV-TTF-1, a kind gift of Dr. R. DiLauro, Stazione
Zoologica "Anton Dohrn" (Naples, Italy). Varying amounts of a
vector plasmid (pBluescript SK; Stratagene, La Jolla, CA) were added to
maintain a constant amount of plasmid in these experiments. After
20-24 h, serum-containing medium was restored. Cells were
harvested 48-72 h after transfection and were assayed for both CAT
and
-galactosidase activity, as described previously (37). CAT
activity per tissue culture dish was divided by
-galactosidase
activity to normalize for efficiency of transfection. SEs were
calculated, and significant changes were evaluated using Student's
t-test (Sigma Plot; Jandel Scientific, Corte Madera, CA).
Oligonucleotides. Oligonucleotides
were obtained from the Cancer Center Nucleic Acid Facility at the
University of Pennsylvania (Philadelphia, PA). Annealing of
single-stranded oligonucleotides was performed by mixing 1 mM solutions
of complementary oligonucleotides in 10 mM
Tris · HCl, 100 mM NaCl, and 1 mM EDTA (pH 8.0),
heating to boiling, and allowing the entire bath to cool to room
temperature. Oligonucleotides were end labeled with
[-32P]ATP (NEN Life
Sciences, Boston, MA) using T4 polynucleotide kinase (New England
Biolabs, Beverly, MA) and were purified through Sephadex G50 or G25
columns (Sigma, St. Louis, MO).
The sequence of the double-stranded oligonucleotide corresponding to
nucleotides 95 to
77 of the SP-A
90
region (Fig. 1, underlined sequence) was
5'-CTTCCTGCCCGGCCCTCCT-3'. The sequence of the mutated
oligonucleotide that was used as a competitor against the
SP-A
90 oligonucleotide was
5'-CTTAAGTCCCGGCCCTCCT-3'. These oligonucleotides had an
extra overhanging base on either side to facilitate labeling, a G on
the 5'-end of the sense strand and a C on the 5'-end of the
antisense strand. The oligonucleotide from
112 to
89 of
the SP-B gene that contained the TTF-1 binding site, a kind of gift of
Dr. A. S. Kumar, Children's Hospital of Philadelphia, had the sequence
5'-GCACCTGGAGGGCTCTTCAGAGC-3'.
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Site-directed mutagenesis. Nucleotides
underlined in Table 1 were
mutated in the 163 SP-A-CAT plasmid with the
Clontech Transformer Mutagenesis kit (Clontech, Palo Alto, CA). Inserts from mutated plasmids were sequenced to confirm that the appropriate bases had been changed and that only the targeted nucleotides had been
mutated. The inserts were then recloned into pCAT-Basic.
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Electrophoretic mobility shift analysis. Nuclear extracts were made as described by Lavery and Schibler (25). Protein concentration of extracts was determined by the Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA). Binding of nuclear proteins to labeled oligonucleotides for electrophoretic mobility shift analysis was performed as described (24). About 10 fmol of labeled oligonucleotide were incubated at 0°C for 30 min with 5 µg of nuclear protein and 0.25 µg of poly(dI-dC) · (dI-dC) (Pharmacia LKB, Uppsala, Sweden) in buffer containing 10 mM Tris · HCl (pH 7.5), 50 mM NaCl, 5% glycerol, 1 mM EDTA, and 1 mM dithiothreitol. Excess unlabeled DNA was added for competition experiments. Samples were applied to nondenaturing 6% polyacrylamide gels in Tris-borate-EDTA buffer. The gels were analyzed by autoradiography. Quantification of signals was performed with an Ambis 4000 imager (Scanalytics, Billerica, MA).
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RESULTS |
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Deletion analysis reveals two active regions in the
promoter of the rat SP-A gene. A short, 163-bp region
of DNA located upstream from the start of transcription of the rat SP-A
gene promotes expression of the reporter gene encoding CAT (163
SP-A-CAT) in the lung cell line NCI-H441, which expresses SP-A, and, to
a lesser extent, in the uterine cervical cell line HeLa, which does not express SP-A (37). To localize the SP-A promoter more precisely, we
used an exonuclease to shorten the 5'-end of the original
construct (Fig. 1). We examined the residual promoter activities of
several deleted constructs by transient transfections in two lung cell lines, NCI-H441, derived from a Clara cell, and MLE-15, derived from a
type II cell.
Regions required for promoter activity differed in the two cell lines.
Promoter activity in transfected NCI-H441 cells was unaffected by
deletion of sequences between 163 and
95 bp. Further deletion to nucleo-tide
75, however, did reduce gene expression by ~50% (Fig. 2), suggesting that the
region between
95 and
75 bp (a region we will call
SP-A
90; see Fig. 1) facilitates transcription. In
contrast, in transfected MLE-15 cells, CAT expression was reduced to
~50% of that of the full-length SP-A-CAT construct when the
nucleotides between
163 and
132 bp were deleted (Fig. 2).
This region in the mouse SP-A gene contains several sequences that
correspond to the recognition element for TTF-1. One of these sites,
which has been shown to be required for full promoter activity of the
mouse SP-A gene in MLE-15 cells (4), is fully conserved in the rat SP-A
gene (Fig. 1). Further deletion to
75 bp, removing the
SP-A
90 region active in NCI-H441 cells, did not
further reduce the promoter activity in MLE-15 cells.
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Mutation of nucleotides within the
SP-A90 region inhibits promoter activity in both
lung cell lines. The deletion analysis suggested that
the SP-A
90 region influenced expression in NCI-H441
cells. In the constructs examined, however, both
SP-A
90 and the TTF-1 recognition element were
deleted. To measure the effect of the SP-A
90 region
alone, we inactivated the region by site-directed mutagenesis. First,
we compared the sequences of the SP-A genes of several species in
regions that correspond to the SP-A
90 region of the
rat gene (Table 1). Four nucleotides were
identical among the species examined except for the rabbit gene, which
differed in one base.
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We mutated the rat 163 SP-A-CAT construct at the four highly
conserved bases (Table 1) and examined the mutant promoter's activity
in the cell lines (Table 2). The mutated SP-A promoter had only about
one-half of the activity of the wild type in both lung cell lines,
indicating that the SP-A
90 region plays a role in
expression of the SP-A gene in both cell lines.
Oligonucleotides corresponding to the
SP-A90 region are recognized by nuclear
proteins. Electrophoretic mobility shift assays were
performed to measure binding of nuclear proteins to the
SP-A
90 region of the rat gene. At least one major
band (resulting from electrophoretic shift of the radiolabeled
SP-A
90 oligonucleotide) was seen after incubation
with nuclear proteins from adult or fetal (21 days of gestation) rat
lung or liver (Fig. 3A).
SP-A
90 binding was also detected in the cell lines used to examine promoter activity, NCI-H441 and MLE-15 (Fig.
3B). The intensity of the major
bands was greatly reduced by the addition of 100-fold excess unlabeled
SP-A
90, suggesting that the binding wasspecific. The
intensity of these bands was not reduced as much when excess unlabeled
mutant SP-A
90 was used. This indicated
that the four conserved nucleotides in the SP-A promoter are required
for efficient binding of these nuclear proteins. Occasionally, a new
band appeared when excess unlabeled mutant SP-A
90
was added to the binding reaction (e.g., fetal rat lung; Fig. 3).
Perhaps the protein(s) responsible for this shift recognized the four
conserved nucleotides in SP-A
90, but their binding
could not be detected unless other proteins, which bind nearby and
block access of these proteins, were competed off. An unrelated
oligonucleotide did not block DNA binding by proteins in the rat lung
extract (data not shown).
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The mobility of the major band differed depending on the source of the nuclear extract. The position of the major fetal and adult lung bands is shown in Fig. 3. The major band from the adult rat lung and liver extracts was larger than the major band from the fetal lung extract (Fig. 3A). Fetal liver extract displayed several bands of different sizes. The cell lines all displayed shifted bands of even larger size, similar in mobility to a minor band from the fetal rat lung (Fig. 3B).
Protein recognition of the SP-A90
oligonucleotide changes during lung development.
Recognition of the SP-A
90 oligonucleotide by rat
nuclear proteins was detected as early as fetal day
19 (Fig. 4). . The larger
band was first clearly seen in nuclear extracts from newborn rats. Both
sizes were still present in extracts from 5-day-old pups, with the
larger one more prominent. Only the larger complex was apparent in the
adult rat lung.
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Slower-migrating bands also appeared. Their intensity relative to the marked bands varied (compare Figs. 3 and 4). The larger complexes could be multimers of the smaller proteins.
Different levels and activity of TTF-1 in the two cell
lines. The second site in the rat SP-A promoter found
to affect transcription (163 to
133 bp) corresponds to a
region of TTF-1 recognition elements in the mouse SP-A gene (4).
Cotransfection experiments were used to examine the effects of TTF-1 on
expression of
163 SP-A-CAT in the cell lines. In NCI-H441 cells,
transactivation of
163 SP-A-CAT by TTF-1 was observed in a
dose-dependent fashion (Fig.
5A),
even though deletion of the TTF-1 recognition element did not affect
promoter activity in these cells. In contrast, in MLE-15 cells,
cotransfection of the TTF-1 expression plasmid with
163 SP-A-CAT
did not lead to dose-dependent activation, even though deletion of the
TTF-1 recognition element did affect promoter activity. As expected,
there was no significant increase in expression of the
132
SP-A-CAT construct (which lacks the TTF-1 binding sites) when the TTF-1
expression plasmid was cotransfected (Fig. 5,
A and
B).
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Both MLE-15 cells (4) and NCI-H441 cells (22) have been shown to
contain TTF-1. A possible explanation for the difference in
transactivation of 163 SP-A-CAT by TTF-1 seen in the cell lines
is that the NCI-H441 cells contain lower levels of endogenous active
TTF-1 than the MLE-15 cells. To evaluate this possibility, we utilized
an oligonucleotide containing the proximal TTF-1 site of the human SP-B
gene (
112 to
89 bp; see Ref. 22) as a probe in an
electrophoretic mobility shift assay with nuclear extracts of the two
cell lines. A representative assay is shown in Fig. 6. Although TTF-1 was detectable in nuclear
extracts of NCI-H441 cells, as noted previously (22), there was about
threefold more active TTF-1 in nuclei of MLE-15 cells than in those of
NCI-H441 cells. Because there was more protein from the mouse cell line binding to the human sequence than from the human cell line, it is
unlikely that this result is due to species differences. It is possible
that the level of TTF-1 in NCI-H441 cells is below the threshold needed
to facilitate expression of the SP-A gene.
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DISCUSSION |
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The data presented here demonstrate that the proximal promoter of the
rat SP-A gene contains at least two functional regions. One of the
regions, which we have designated SP-A90, lies between nucleotides
95 and
75. Its mutation results in
loss of ~50% of the transcriptional activity in a Clara cell line
and a type II cell line. Therefore, SP-A
90 appears
to be required for full expression of the SP-A gene in lung cells.
In the Clara cell line NCI-H441, transcriptional activity is not
affected by 5' deletion until the SP-A90
region is removed. Its deletion reduces activity by ~50%. In the
type II cell line MLE-15, ~50% of the promoter activity is lost when
nucleotides further upstream (
163 to
133) are deleted.
This second region contains a recognition element for the transcription
factor TTF-1. TTF-1 activity was previously identified in the mouse
SP-A promoter (4). Further deletion through SP-A
90
does not further reduce promoter activity in MLE-15 cells. Inactivation
of SP-A
90 alone by mutagenesis, however, does reduce
promoter activity. Thus, in MLE-15 cells, either deletion of the TTF-1
recognition element or mutation of SP-A
90 reduces
SP-A promoter activity by one-half, but deletion of the two together
does not further reduce activity. These results suggest that both
regions together are required for the activity, which is lost upon
inactivation of either one.
The SP-A90 region has the ability to bind rat
nuclear proteins. The same mutation that reduces function also
interferes with the ability of the region to bind proteins. The
identity of the bound protein(s) is unknown at this time because the
sequence of the SP-A
90 region does not match
extensively with any of the transcription factor binding sites in
databases examined. Protein binding is not tissue specific, with
proteins from both liver and lung nuclear extracts binding. This is in
accord with our previous results (37), which suggest that the proximal
SP-A promoter is capable of directing expression in both lung and
nonlung cell types.
Binding of nuclear proteins to the SP-A90 region
changes during late fetal and neonatal development. In lung, a clear change in the mobility of the protein-DNA complexes on nondenaturing electrophoretic gels occurs between the fetal and adult stages of
development. The larger-size complex seen in the adult does not appear
before fetal day 20, whereas SP-A mRNA
is detectable by embryonic day 18 by
Northern analysis (10) and by day 13 by PCR analysis (43). The alterations in control of the gene's expression suggested by the changes in the protein binding to SP-A
90 would, thus, not influence the onset of
transcription of the SP-A gene. Alterations in control of the gene's
expression could represent an increase in transcription or changes
occurring in control of the surfactant system at birth, when breathing
is initiated.
Shifts of radiolabeled SP-A90 to different
electrophoretic mobilities could signify several differences between
the fetal and adult lung binding proteins. For example, covalent
modification of the DNA binding protein(s) could vary, the size of the
same gene product might vary due to alternative splicing or
proteolysis, or entirely different SP-A
90 binding
proteins might exist in the fetal and adult lungs. The major
SP-A
90 binding complexes from the cell lines have a
slower mobility than the major complex from either fetal or adult lung,
although they have a mobility similar to a minor fetal lung
SP-A
90 binding complex. The increased size of the
major complexes from the cell lines could be due to multimerization or
to any of the possibilities mentioned above. Differences in
SP-A
90 binding between lung cell lines and the lung
itself could mean that observations made about the cells only partially
reflect the in vivo situation.
The second active region in the SP-A promoter (from 163 to
133 bp) contains binding sites for the transcription factor
TTF-1 and has different activities in the two cell lines examined.
Deleting this region reduces expression from the rat SP-A promoter in
MLE-15 cells, as expected from the results with the mouse SP-A promoter (4). However, deleting the TTF-1 region has no discernible effect on
SP-A promoter-directed expression in the NCI-H441 cell line. Similar
results were obtained in these cells with the mouse promoter (4). Our
experiments suggest that, in fact, the rat SP-A promoter can be
activated by TTF-1 in NCI-H441 cells but that these cells contain less
of the active TTF-1 than MLE-15 cells. Because TTF-1 has been shown to
activate the SP-B promoter (47), this may account for the finding that
there are higher levels of SP-B mRNA in MLE cells than in NCI-H441
cells (unpublished observations). On the other hand, because MLE-15
cells express much less SP-A mRNA than NCI-H441 cells, these data also
suggest that TTF-1 alone is not sufficient for full expression of the SP-A gene.
Samples examined in this work are derived from different species. The cells come from a human and a mouse, whereas the SP-A gene comes from a rat. The SP-A promoters of these species, however, are highly conserved (37). Therefore, transcription factors common to all three species, such as TTF-1, are likely to activate each of these promoters. The patterns of SP-A gene expression are also quite similar. SP-A is expressed in type II cells, starting late in fetal gestation in the three species (8, 14, 19). In the rat (15, 39) and mouse (20), Clara cells also produce SP-A. Several investigators have observed SP-A mRNA in human Clara cells (2, 17), although its absence has also been reported (32). Because of the general similarity, we have used cell lines from a mouse and human to study the rat gene.
Two other laboratories have studied transcriptional control of the rat SP-A gene. Both have used in vitro transcription, but they reached different conclusions. Lacaze-Masmonteil et al. (23) found that a fragment containing 212 nucleotides before the start of transcription was transcribed in fetal lung nuclear extracts but not in adult liver nuclear extracts. However, Stuempfle et al. (38) found that several constructs containing between 1132 and 134 nucleotides before the start of transcription were all capable of directing in vitro transcription in both lung and liver nuclear extracts.
Control of transcription of SP-A genes from other species has been
studied in a number of laboratories. As stated above, TTF-1 sites have
been shown to affect the promoter activity of the mouse gene in MLE-15
cells (4). Mutation of TTF-1 binding sites in the baboon SP-A2 gene was
shown to affect both basal and cAMP-stimulated expression in type II
cells isolated from human fetal lung (28). Other transcription factors
have also been implicated in SP-A gene expression. Gao et al. (11)
identified a proximal binding element containing a palindrome at
position 81 bp (sequence CTCGTG) in the rabbit SP-A gene. This
sequence, called an E box, was shown to bind the transcription factor
upstream stimulatory factor-1 (12). Deletion of this region
substantially reduced both basal and cAMP-enhanced expression of rabbit
SP-A reporter gene constructs in type II cells. The rat gene has a
related sequence at
81 bp (CTCCTG), within the region of
SP-A
90. However, the palindrome is not conserved,
and the effect of deleting the SP-A
90 region can be
mimicked by mutating four base pairs outside this box (Table 1). Thus
it is unlikely that this sequence is functional in the rat SP-A gene.
In summary, the SP-A promoter appears to be complex, with a number of
different DNA domains binding a variety of transcription factors. We
have identified a novel domain, SP-A90, affecting promoter activity in the rat SP-A gene. This region is recognized by
nuclear proteins. The SP-A
90 region may interact
with another region of the promoter containing recognition elements for
the transcription factor TTF-1. We have found that these elements may
play different roles in the regulation of SP-A gene expression at
different stages of development and in different cell types.
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ACKNOWLEDGEMENTS |
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We thank Dr. Jeffrey Whitsett for the MLE-15 cell line, Dr. Robert DiLauro for the CMV-TTF-1 plasmid, Dr. A. Suresh Kumar for the TTF-1 binding oligonucleotide, and Colleen Campbell for proofreading the manuscript.
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FOOTNOTES |
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This work was supported by an American Lung Association grant (E. Rosenberg) and by National Heart, Lung, and Blood Institute Grants HL-19737, SCOR 1-P50 HL-56401, and HL-53566 (S. I. Feinstein). F. Li and C. I. Smith were supported by National Heart, Lung, and Blood Institute Institutional National Research Service Award HL-07027.
This work was presented in part at the American Lung Association/American Thoracic Society International Conferences in Seattle, WA, May 1995, San Francisco, CA, May 1997, and Chicago, IL, April 1998.
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 correspondence: S. I. Feinstein, Institute for Environmental Medicine, Rm. 1, John Morgan Bldg., School of Medicine, Univ. of Pennsylvania, Philadelphia, PA 19104-6068 (E-mail: sif{at}mail.med.upenn.edu).
Received 15 June 1998; accepted in final form 4 March 1999.
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REFERENCES |
---|
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---|
1.
Ausubel, F. M.,
R. Brent,
R. E. Kingston,
D. D. Moore,
J. G. Seidman,
J. A. Smith,
and
K. Struhl.
Current Protocols in Molecular Biology. New York: Wiley, 1994.
2.
Auten, R. L.,
R. H. Watkins,
D. L. Shapiro,
and
S. Horowitz.
Surfactant apoprotein A (SP-A) is synthesized in airway cells.
Am. J. Respir. Cell Mol. Biol.
3:
491-496,
1990[Medline].
3.
Ballard, P. L.,
S. Hawgood,
H. Liley,
G. Wellenstein,
L. W. Gonzales,
B. Benson,
B. Cordell,
and
R. T. White.
Regulation of pulmonary surfactant apoprotein SP 28-36 gene in fetal human lung.
Proc. Natl. Acad. Sci. USA
83:
9527-9531,
1986[Abstract].
4.
Bruno, M. D.,
R. J. Bohinski,
K. M. Huelsman,
J. A. Whitsett,
and
T. R Korfhagen.
Lung cell-specific expression of the murine surfactant protein A (SP-A) gene is mediated by interactions between the SP-A promoter and thyroid transcription factor-1.
J. Biol. Chem.
270:
6531-6536,
1995
5.
Chen, Q.,
V. Boggaram,
and
C. R. Mendelson.
Rabbit lung surfactant protein A gene: identification of a lung-specific DNase I hypersensitive site.
Am. J. Physiol.
262 (Lung Cell. Mol. Physiol. 6):
L662-L671,
1992
6.
Chung, J.,
S. H. Yu,
J. A. Whitsett,
P. G. Harding,
and
F. Possmayer.
Effect of surfactant-associated protein-A (SP-A) on the activity of lipid extract surfactant.
Biochim. Biophys. Acta
1002:
348-358,
1989[Medline].
7.
Crouch, E. C.
Collectins and pulmonary host defense.
Am. J. Respir. Cell Mol. Biol.
19:
177-201,
1998
8.
Endo, H.,
and
T. Oka.
An immunohistochemical study of bronchial cells producing surfactant protein A in the developing human fetal lung.
Early Hum. Dev.
25:
149-156,
1991[Medline].
9.
Farrell, P. M.,
and
M. E Avery.
Hyaline membrane disease.
Am. Rev. Respir. Dis.
111:
657-688,
1975[Medline].
10.
Fisher, J. H.,
P. A. Emrie,
J. Shannon,
K. Sano,
B. Hattler,
and
R. J. Mason.
Rat pulmonary surfactant protein A is expressed as two differently sized mRNA species which arise from differential polyadenylation of one transcript.
Biochim. Biophys. Acta
950:
338-345,
1989.
11.
Gao, E.,
J. L. Alcorn,
and
C. R. Mendelson.
Identification of enhancers in the 5'-flanking region of the rabbit surfactant protein A (SP-A) gene and characterization of their binding proteins.
J. Biol. Chem.
268:
19697-19709,
1993
12.
Gao, E.,
Y. Wang,
J. L. Alcorn,
and
C. R. Mendelson.
The basic helix-loop-helix-zipper transcription factor USF-1 regulates expression of the surfactant protein-A gene.
J. Biol. Chem.
272:
23398-23406,
1997
13.
Horowitz, S.,
R. H. Watkins,
R. L. Auten, Jr.,
C. E. Mercier,
and
E. R. Cheng.
Differential accumulation of surfactant protein A, B, and C mRNAs in two epithelial cell types of hyperoxic lung.
Am. J. Respir. Cell Mol. Biol.
5:
511-515,
1991[Medline].
14.
Jaskoll, T. F.,
D. Phelps,
H. W. Taeusch,
B. Smith,
and
H. C. Slavkin.
Localization of pulmonary surfactant protein during mouse lung development.
Dev. Biol.
106:
256-261,
1984[Medline].
15.
Kalina, M.,
R. J. Mason,
and
J. M. Shannon.
Surfactant protein C is expressed in alveolar type II cells but not in Clara cells of rat lung.
Am. J. Respir. Cell Mol. Biol.
6:
594-600,
1992[Medline].
16.
Katyal, S. L.,
G. Singh,
and
J. Locker.
Characterization of a second human pulmonary surfactant-associated protein SP-A gene.
Am. J. Respir. Cell Mol. Biol.
6:
446-452,
1992[Medline].
17.
Khoor, A.,
M. E. Gray,
W. M. Hull,
J. A. Whitsett,
and
M. T. Stahlman.
Developmental expression of SP-A and SP-A mRNA in the proximal and distal respiratory epithelium in human fetus and newborn.
J. Histochem. Cytochem.
41:
1311-1309,
1993
18.
King, R. J.
Pulmonary surfactant.
J. Appl. Physiol.
53:
1-8,
1982
19.
Kohri, T.,
K. Sakai,
T. Mizunuma,
and
Y. Kishino.
Levels of pulmonary surfactant protein A in fetal lung and amniotic fluid from protein malnourished pregnant rats.
J. Nutr. Sci. Vitaminol. (Tokyo)
42:
209-218,
1996[Medline].
20.
Korfhagen, T. R.,
M. D. Bruno,
S. W. Glasser,
P. J. Ciraolo,
J. A. Whitsett,
D. L. Lattier,
K. A. Wikenheiser,
and
J. C. Clark.
Murine pulmonary surfactant SP-A gene: cloning, sequence, and transcriptional activity.
Am. J. Physiol.
263 (Lung Cell. Mol. Physiol. 7):
L546-L555,
1992
21.
Korfhagen, T. R.,
M. D. Bruno,
G. F. Ross,
K. M. Huelsman,
M. Ikegami,
A. H. Jobe,
S. E. Wert,
B. R. Stripp,
R. E. Morris,
S. W. Glasser,
C. J. Bachurski,
H. Iwamoto,
and
J. A. Whitsett.
Altered surfactant function and structure in SP-A gene targeted mice.
Proc. Natl. Acad. Sci. USA
93:
9594-9599,
1996
22.
Kumar, A. S.,
V. C. Venkatesh,
B. C. Planer,
S. I. Feinstein,
and
P. L. Ballard.
Phorbol ester down-regulation of lung surfactant protein B gene expression by cytoplasmic trapping of thyroid transcription factor-1 and hepatocyte nuclear factor 3.
J. Biol. Chem.
272:
20764-20773,
1997
23.
Lacaze-Masmonteil, T.,
C. Fraslon,
J. Bourbon,
and
A. Kahn.
Characterization of the rat pulmonary surfactant protein A promoter.
Eur. J. Biochem.
206:
613-623,
1992[Abstract].
24.
Ladias, J. A.,
and
S. K. Karathanasis.
Regulation of the apolipoprotein AI gene by ARP-1, a novel member of the steroid receptor superfamily.
Science
251:
561-565,
1991[Medline].
25.
Lavery, D. J.,
and
U. Schibler.
Circadian transcription of the cholesterol 7 hydroxylase gene may involve the liver-enriched bZIP protein DBP.
Genes Dev.
7:
1871-1884,
1993[Abstract].
26.
LeVine, A. M.,
M. D. Bruno,
K. M. Huelsman,
G. F. Ross,
J. A. Whitsett,
and
T. R. Korfhagen.
Surfactant protein A-deficient mice are susceptible to group B streptococcal infection.
J. Immunol.
158:
4336-4340,
1997[Abstract].
27.
LeVine, A. M.,
M. Bruno,
M. J. Whitsett,
and
T. Korfhagen.
Surfactant protein A in pulmonary host defense against bacterial pathogens in vivo (Abstract).
Am. J. Respir. Crit. Care Med.
155:
A214,
1997.
28.
Li, J.,
E. Gao,
and
C. R. Mendelson.
Cyclic AMP-responsive expression of surfactant protein-A gene is mediated by increased DNA binding and transcriptional activity of thyroid transcription factor-1.
J. Biol. Chem.
273:
4592-4600,
1998
29.
Mendelson, C. R.,
and
V. Boggaram.
Hormonal control of the surfactant system in fetal lung.
Annu. Rev. Physiol.
53:
415-440,
1991[Medline].
30.
O'Reilly, M. A.,
A. F. Gazdar,
R. E. Morris,
and
J. A. Whitsett.
Differential effects of glucocorticoid on expression of surfactant proteins in a human lung adenocarcinoma cell line.
Biochim. Biophys. Acta
970:
194-204,
1988[Medline].
31.
Perelman, R. H.,
P. M. Farrell,
M. J. Engle,
and
J. W. Kemnitz.
Developmental aspects of lung lipids.
Annu. Rev. Physiol.
47:
803-822,
1985[Medline].
32.
Phelps, D. S.,
and
J. Floros.
Localization of surfactant protein synthesis in human lung by in situ hybridization.
Am. Rev. Respir. Dis.
137:
939-942,
1988[Medline].
33.
Rice, W. R.,
G. F. Ross,
F. M. Singleton,
S. Dingle,
and
J. A. Whitsett.
Surfactant-associated protein inhibits phospholipid secretion from type II cells.
J. Appl. Physiol.
63:
692-698,
1987
34.
Rubio, S.,
T. Lacaze-Masmonteil,
B. Chailley-Heu,
A. Kahn,
J. R. Bourbon,
and
R. Ducroc.
Pulmonary surfactant protein A (SP-A) is expressed by epithelial cells of small and large intestine.
J. Biol. Chem.
270:
12162-12169,
1995
35.
Saitoh, H.,
H. Okayama,
S. Shimura,
T. Fushimi,
T. Masuda,
and
K. Shirato.
Surfactant protein A2 gene expression by human airway submucosal gland cells.
Am. J. Respir. Cell Mol. Biol.
19:
202-209,
1998
36.
Sawaya, P. L.,
B. R. Stripp,
J. A. Whitsett,
and
D. S. Luse.
The lung specific CC10 gene is regulated by transcription factors from the AP-1, octamer and hepatocyte nuclear factor 3 families.
Mol. Cell. Biol.
13:
3860-3871,
1993[Abstract].
37.
Smith, C. I.,
E. Rosenberg,
S. R. Reisher,
F. Li,
P. Kefalides,
A. B. Fisher,
and
S. I. Feinstein.
Sequence of the rat surfactant protein A gene and functional mapping of its upstream region.
Am. J. Physiol.
269 (Lung Cell. Mol. Physiol. 13):
L603-L612,
1995
38.
Stuempfle, K. J.,
M. Koptides,
P. G. Quinn,
and
J. Floros.
In vitro analysis of rat surfactant protein A gene expression.
Am. J. Physiol.
270 (Lung Cell. Mol. Physiol. 14):
L504-L516,
1996
39.
Sugahara, K.,
K. Iyama,
K. Sano,
and
T. Morioka.
Differential expressions of surfactant protein SP-A, SP-B, and SP-C mRNAs in rats with streptozotocin-induced diabetes demonstrated by in situ hybridization.
Am. J. Respir. Cell Mol. Biol.
11:
397-404,
1994[Abstract].
40.
Veldhuizen, R. A.,
L. J. Yao,
S. A. Hearn,
F. Possmayer,
and
J. F. Lewis.
Surfactant-associated protein A is important for maintaining surfactant large-aggregate forms during surface-area cycling.
Biochem. J.
313:
835-840,
1996[Medline].
41.
Venkatesh, V. C.,
B. C. Planer,
M. Schwartz,
J. N. Vanderbilt,
R. T. White,
and
P. L. Ballard.
Characterization of the promoter of human pulmonary surfactant protein B gene.
Am. J. Physiol.
268 (Lung Cell. Mol. Physiol. 12):
L674-L682,
1995
42.
Voorhout, W. F.,
T. Veendendaal,
H. P. Haagsman,
A. J. Verkleij,
L. M. van Golde,
and
H. J. Geuze.
Surfactant protein A is localized at the corners of the pulmonary tubular myelin lattice.
J. Histochem. Cytochem.
39:
1331-1336,
1991[Abstract].
43.
Wang, J.,
P. Souza,
M. Kuliszewski,
A. K. Tanswell,
and
M. Post.
Expression of surfactant proteins in embryonic rat lung.
Am. J. Respir. Cell Mol. Biol.
10:
222-229,
1994[Abstract].
44.
Weaver, T. E.,
and
J. A. Whitsett.
Function and regulation of expression of pulmonary surfactant-associated proteins.
Biochem. J.
273:
249-264,
1991[Medline].
45.
Wikenheiser, K. A.,
D. K. Vorbroker,
W. R. Rice,
J. C. Clark,
C. J. Bachurski,
H. K. Oie,
and
J. A. Whitsett.
Production of immortalized distal respiratory epithelial cell lines from surfactant protein C/simian virus 40 large tumor antigen transgenic mice.
Proc. Natl. Acad. Sci. USA
90:
11029-11033,
1993[Abstract].
46.
Wright, J. R.,
R. E. Wager,
S. Hawgood,
L. Dobbs,
and
J. A. Clements.
Surfactant apoprotein Mr=26,000-36,000 enhances uptake of liposomes by type II cells.
J. Biol. Chem.
262:
2888-2894,
1987
47.
Yan, C.,
Z. Sever,
and
J. A. Whitsett.
Upstream enhancer activity in the human surfactant protein B gene is mediated by thyroid transcription factor 1.
J. Biol. Chem.
270:
24852-24857,
1995
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