Transcriptional activation and protein binding by two regions of the rat surfactant protein A promoter

Elizabeth Rosenberg1, Feng Li2, Candyce I. Smith3, Samuel R. Reisher1, and Sheldon I. Feinstein1

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


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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-A-90. 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.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta -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 beta -galactosidase activity, as described previously (37). CAT activity per tissue culture dish was divided by beta -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 [gamma -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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Fig. 1.   Surfactant protein (SP) A promoter sequence. Region from 163 to 74 bases upstream from the transcriptional start (-163 to -74) is shown. Positions of the first nucleotide of some of the key deletions used to map the promoter are indicated (-132, -95, and -75). The last digit of the number is positioned over the corresponding nucleotide. The thyroid transcription factor (TTF)-1 site corresponding to the one shown to affect expression of the mouse SP-A promoter in MLE-15 cells (4) is shown in bold italics. The region (SP-A-90) that reduces expression in NCI-H441 cells when deleted is underlined. The four bases that were mutated are shown in bold type.

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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Table 1.   Mutation of four residues in the SP-A gene conserved among species

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).


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 2.   Deletion analysis in two cell lines. NCI-H441 cells (filled bars) and MLE-15 cells (hatched bars) were transfected with chloramphenicol acetyltransferase (CAT) plasmids containing DNA from the promoter of the rat SP-A gene. The 3' terminus of all constructs was +34 bp. CAT activities were measured 48-72 h after transfection. Activity of each SP-A-CAT construct was corrected for transfection efficiency by the use of a plasmid expressing beta -galactosidase. The results were normalized to the -163 construct, which has been set to 100%. Values are means ± SE; n >=  4 independent determinations. * P <=  0.05 vs. SP-A-163-CAT constructs measured in the same cell line.

Mutation of nucleotides within the SP-A-90 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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Table 2.   Activity of the SP-A promoter mutated at four residues conserved among species

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-A-90 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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Fig. 3.   Nuclear proteins bind to the rat SP-A-90 region. An end-labeled oligonucleotide (oligo) representing the rat SP-A-90 region was allowed to bind nuclear extracts from different sources and was subjected to electrophoresis on 6% polyacrylamide gels. Nuclear extracts from adult and fetal rat lungs and livers (A) and adult and fetal lungs as well as lung cell lines (B) are shown. Arrows indicate major shifted bands in adult and fetal lungs. Arrowhead indicates shifted band in cell lines. In indicated lanes, binding was competed with a ×100 molar excess of unlabeled wild-type oligonucleotide or unlabeled mutant oligonucleotide, in which the four bases underlined in Table 1 (CCTG) were mutated (to AAGT). Note that most of the nuclear proteins that bind to SP-A-90 can be competed with an excess of the wild-type but not of the mutant oligonucleotide.

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-A-90 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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Fig. 4.   Developmental changes in proteins binding to the rat SP-A-90 region. An end-labeled oligonucleotide representing the rat SP-A-90 region was allowed to bind nuclear extracts from fetal rat lungs of different developmental times during gestation and after birth. The reactions were subjected to electrophoresis on a 6% polyacrylamide gel. Arrows indicate major shifted bands in adult and fetal lungs.

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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Fig. 5.   Effect of TTF-1 cotransfection on expression of rat SP-A-CAT constructs. NCI-H441 cells (A) and MLE-15 cells (B) were transfected with CAT plasmids containing regions of the SP-A promoter from either -163 (filled bars) or -132 (hatched bars) nucleotides through +34 bp. At the same time, cells were also transfected with the indicated amounts of a TTF-1 expression plasmid. Vector plasmid was added to keep the total amount of DNA constant. CAT activities were measured 48-72 h after transfection. Activity of each SP-A-CAT construct has been corrected for transfection efficiency by the use of a plasmid expressing beta -galactosidase. Results have been normalized to the -163 construct, which has been set to 100%. Values are means ± SE; n = 4 independent determinations. * P <=  0.05 vs. 0 ng TTF-1 plasmid in NCI-H441 cells.

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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Fig. 6.   Electrophoretic mobility shift assays of TTF-1 binding. Radiolabeled oligonucleotide containing the TTF-1 binding site was allowed to bind to 5 µg of nuclear proteins from the indicated cell lines. Results of a representative experiment are shown. Arrow indicates TTF-1-shifted bands.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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-A-90, 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-A-90 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-A-90 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-A-90 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-A-90 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-A-90, 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.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.

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Am J Physiol Lung Cell Mol Physiol 277(1):L134-L141
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society




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