Differential regulation of baboonSP-A1 andSP-A2 genes: structural and functional analysis of 5′-flanking DNA

Jinxing Li, Erwei Gao, Steven R. Seidner, Carole R. Mendelson

Abstract

Surfactant protein (SP) A gene transcription is developmentally regulated and stimulated by hormones and factors that increase intracellular cAMP. The baboon (b) genome contains two highly similarSP-A genes,bSP-A1 andbSP-A2. With the use of a ribonuclease protection assay with gene-specific probes, the two bSP-Agenes were found to be differentially regulated during baboon fetal lung development in that expression of thebSP-A2 gene appeared to be induced to a high level at a later time in gestation than that of thebSP-A1 gene. Both thebSP-A1 andbSP-A2 genes were found to be highly responsive to the inductive effects of cAMP in baboon fetal lung explants in culture. By DNase I footprinting and electrophoretic mobility shift assays with bacterially expressed thyroid transcription factor-1 (TTF-1) and type II cell nuclear extracts, three TTF-1 binding elements were identified within the 255-bp region flanking the 5′-end of each bSP-A gene; however, these differed in position and spacing for the twobSP-A genes. To functionally define the genomic regions that are required for cAMP regulation ofbSP-A gene expression in type II cells, fusion genes composed of various amounts of 5′-flanking DNA from the bSP-A1 andbSP-A2 genes linked to the human growth hormone structural gene as a reporter were transfected into type II cells in primary culture. We found that 255 bp of 5′-flanking DNA, which contain three TTF-1 binding elements, frombSP-A1 andbSP-A2 genes were sufficient to mediate high basal and cAMP-inducible expression in type II cells. We also observed that there were no obvious differences in the magnitude of the responses of these fusion genes to cAMP treatment.

  • surfactant protein A
  • deoxyribonucleic acid
  • fetal lung
  • thyroid transcription factor-1
  • ribonuclease protection assay

pulmonary surfactant, a lipoprotein synthesized exclusively by alveolar type II cells, acts to reduce alveolar surface tension, thereby preventing alveolar collapse on the exhalation of air. Four lung-specific proteins have been found to be associated with surfactant: surfactant protein (SP) A, SP-B, SP-C, and SP-D. These appear to serve differential roles in the reduction of alveolar surface tension, surfactant phospholipid reutilization, and immune defense within the alveolus (15).

Expression of the gene encoding SP-A, the major surfactant protein, is lung specific; SP-A is expressed primarily in alveolar type II cells and, to a lesser extent, in bronchiolar epithelial (Clara) cells (3,38). Expression of the SP-A gene is also developmentally regulated; gene transcription is initiated in the fetal lung only after ∼70% of gestation is completed and reaches maximum levels toward term (5). Various hormonal factors have been found to regulate SP-A gene expression; cAMP and glucocorticoids are reported to have major regulatory effects (24, 28, 31, 32). In studies using rabbit (29), human (32), and baboon (35) fetal lungs in organ culture, SP-A mRNA and protein levels were found to be augmented by cAMP analogs and by agents that increase the levels of intracellular cAMP. cAMP also enhances the rate of type II cell differentiation and enlargement of prealveolar ducts (32). The effects of glucocorticoids onSP-A gene expression are more complex; e.g., in human fetal lung explants, dexamethasone (Dex) acts synergistically with cAMP to increaseSP-A gene transcription. However, Dex causes a dose-dependent decrease in SP-A mRNA stability (6, 7).

SP-A is encoded by a single-copy gene in rabbits (5), rats (11), dogs (4), and mice (19); however, it has been found that the human (16, 26) and the baboon (13) genomes contain two highly similarSP-A genes (SP-A1 andSP-A2). The two human (h)SP-A genes (hSP-A1 andhSP-A2) appear to be differentially regulated during development and by cAMP and glucocorticoids (27). In lung tissue from a 28-wk-gestation neonate, only hSP-A1 mRNA transcripts were detected, whereas in adult human lung tissues, the ratio of hSP-A2 to hSP-A1 mRNA transcripts was found to be 3:1 (27). These preliminary findings suggest that expression of hSP-A1 is initiated earlier in development than that of hSP-A2, whereas hSP-A2 transcripts are more highly expressed postnatally. ThehSP-A2 gene also was found to be more responsive to the inductive effects of cAMP than thehSP-A1 gene (27). In midgestation human fetal lung explants cultured in control medium, the majority of SP-A transcripts were found to be SP-A1; in tissues cultured in the presence of dibutyryl cAMP (DBcAMP), the ratio of SP-A2 to SP-A1 mRNA was increased to levels similar to that in adult lung tissues (27).

Recently, Seidner et al. (35) reported the use of the baboon as a model for study of SP-A gene regulation. In studies using lung tissues of 92- to 140-day gestational age fetal baboons (term 184 days), the effects of cAMP and Dex were highly similar to those observed with lung explants of midgestation human abortuses; i.e., cAMP had a marked stimulatory effect, whereas Dex caused a dose-dependent decrease in SP-A mRNA levels (35). Sequence comparison of DNA upstream of the transcription initiation sites and within the 3′-untranslated regions of the two baboon (b)SP-A(bSP-A1 andbSP-A2) and the twohSP-A genes indicates thatbSP-A1 is more similar tohSP-A1, whereasbSP-A2 is more similar tohSP-A2 (13).

In consideration of the similarities of thehSP-A andbSP-A genes and their hormonal regulation, as well as of the limited availability of third-trimester human fetal lung tissues, in the present study, we used the baboon as a model to study differential regulation of theSP-A1 andSP-A2 genes during development and by cAMP. To begin to understand the mechanisms whereby these twobSP-A genes are differentially regulated, we transfected type II cells in primary culture with fusion genes composed of up to 1,000 bp of 5′-flanking DNA from eachbSP-A gene to compare their capacity to mediate cAMP induction of SP-A promoter activity. We also analyzed the proximal 5′-flanking regions of bothbSP-A genes for binding sites for thyroid transcription factor-1 (TTF-1), a homeodomain transcription factor expressed specifically in the developing thyroid and lung epithelia and in restricted areas of the developing brain (14, 21). TTF-1 is required for organogenesis of the thyroid, lung, and anterior pituitary (18) and mediates expression of thyroid-specific genes (10,12, 14, 36). It has been reported that TTF-1 binds to and transactivates the SP-A, SP-B, SP-C, and Clara cell-specific protein (Clara cell 10-kDa secretory protein) gene promoters (8, 9, 17, 33). Recently, Li et al. (23) found that cAMP-responsive expression of thebSP-A gene is mediated by increased phosphorylation and binding of TTF-1.

MATERIALS AND METHODS

Culture of fetal lung explants, isolation of type II pneumonocytes, and preparation of type II cell nuclear extracts. Fetal baboons were delivered by hysterotomy after time-dated pregnancies that were confirmed by fetal morphometrics on maternal ultrasounds at 70 and 100 days gestational age. All studies were approved by the Institutional Animal Care Committee at the Southwest Foundation for Biomedical Research (Dallas, TX) and strictly adhered to the National Research Council’s Guide for Care and Use of Laboratory Animals. Fetuses were delivered at the following gestational ages: 92, 125, 140, 160, and 175 days. Lung tissues were minced into 1- to 2-mm3 fragments and cultured on lens papers supported by stainless steel grids in serum-free Waymouth MB 752/1 medium alone or in medium containing DBcAMP (1 mM) (35, 37). The procedure used to isolate and maintain type II pneumonocytes in primary culture has been previously described (2). Briefly, lung tissues of midgestation human abortuses were maintained in organ culture for 5 days in serum-free Waymouth MB 752/1 medium in the presence of DBcAMP (1 mM) to promote type II cell differentiation (32). The explants were then incubated with collagenase (0.5 mg/ml) to disperse the cells and were subsequently treated with DEAE-dextran, which selectively eliminates fibroblasts. The enriched type II cell suspension was plated onto culture dishes that were coated with an extracellular matrix from Madin-Darby canine kidney (MDCK) cells and cultured in Waymouth MB 752/1 medium containing 1 mM DBcAMP for another 4 days (2). Nuclear extracts were prepared from these isolated type II cells as previously described (22, 23).

RNase protection assay. A 150-bp32P-labeled antisense RNA probe for the bSP-A1 gene was transcribed from +3,678 to +3,827 bp of bSP-A1genomic DNA (13). A 190-bp32P-labeled antisense RNA probe for the bSP-A2 gene was transcribed from +3,644 to +3,833 bp of bSP-A2genomic DNA (13). A 32P-labeled 18S rRNA antisense probe was generated by in vitro transcription of pTRI RNA 18S (Ambion, Austin, TX). To detect the transcripts of thebSP-A1 orbSP-A2 genes, 2.5 μg of total RNA were combined with 2 × 104counts/min (cpm) of the bSP-A1 or bSP-A2 RNA probe. Each reaction mixture also contained 1 × 103 cpm of 18S rRNA probe; 2 μg of unlabeled 18S rRNA probe were added to each reaction to ensure that the probe was in molar excess of 18S rRNA. Reaction mixtures were heated at 95°C for 4 min, followed by incubation at 45°C overnight. Reaction mixtures were then digested with 200 μl of a diluted RNase A-RNase T1 mixture (1:100 dilution; Ambion) and incubated at 37°C for 30 min. Protected RNA probes were then ethanol precipitated, resolved on a 6% denaturing polyacrylamide gel, and detected by autoradiography. The relative amounts of bSP-A mRNA and 18S rRNA were assessed by scanning densitometry. After densitometry, the levels of bSP-A1 and bSP-A2 mRNA transcripts were corrected by normalization to the levels of 18S rRNA.

DNase I footprinting. A 321-bp TTF-1 cDNA fragment containing the entire homeodomain region (TTFHD) was amplified with two primers (5′-TTFHD, 5′-TCCGACGTGAGCAAGAACATG-3′ and 3′-TTFHD, 5′-TCACTGCTGCGCCGCCTTGTC-3′), with the baboon TTF-1 cDNA clone as a template. This DNA fragment was incorporated into the bacterial expression vector pGEX-KG (Pharmacia) in-frame with glutathione S-transferase (GST). The GST-TTFHD polypeptide was prepared from Escherichia coli according to procedures suggested by the manufacturer (Pharmacia). DNA probes for footprinting analysis were prepared by PCR with32P end-labeled synthetic oligonucleotides as primers (34). ThebSP-A1 andbSP-A2 genomic clones were used as templates for the amplification of sequences between base pairs −253 and +40 for bSP-A1 and −255 and +40 for bSP-A2. The DNase I footprinting assays were performed in a 200-μl reaction volume. DNA-binding reactions were carried out in a mixture containing 10 mM Tris ⋅ HCl (pH 8.0), 5 mM MgCl2, 1 mM CaCl2, 2 mM dithiothreitol, 50 μg/ml of BSA, 2 μg/ml of calf thymus DNA, and 100 mM KCl. Bacterially expressed proteins (either GST or GST-TTFHD peptide) were incubated with 20,000 cpm of radiolabeled DNA fragment. After 30 min of incubation at room temperature, the reaction mixtures were digested with DNase I for 2 min and rapidly stopped by the addition of 700 μl of DNase I stop solution containing 645 μl of 100% ethanol, 5 μg of tRNA, and 50 μl of saturated ammonium acetate. The reaction products were fractionated on 6% polyacrylamide-7 M urea sequencing gels; the sequencing of the probe was performed as described by Maxam and Gilbert (25), and a sample of the sequencing reaction was loaded adjacent to the samples analyzed by DNase I footprinting.

Electrophoretic mobility shift assays.Oligonucleotides were end labeled with T4 polynucleotide kinase and [γ-32P]ATP. Bacterially expressed GST-TTFHDpolypeptide (2 μg) or alveolar type II cell nuclear extracts (10 μg) were incubated at room temperature for 30 min in binding buffer (20 mM HEPES, pH 7.6, 100 mM KCl, 0.2 mM EDTA, 0.5 mM dithiothreitol, and 20% glycerol) with a radiolabeled DNA probe (10,000 cpm); 2 μg of poly(dI-dC) ⋅ poly(dI-dC) were simultaneously added as a nonspecific competitor. Protein-DNA complexes were resolved on 5% nondenaturing polyacrylamide gels and visualized by autoradiography. For DNA competition electrophoretic mobility shift assay (EMSA), nonradiolabeled double-stranded oligonucleotides were added simultaneously with labeled probe. Supershift EMSA was performed by adding 1 μl of antiserum to the binding reaction, followed by a 30-min incubation at room temperature before electrophoresis.

Construction of bSP-A-human growth hormone fusion genes and preparation of recombinant adenoviruses.Fusion genes composed of 50, 253 or 255, and 1,068 or 986 bp of DNA flanking the 5′-ends plus 40 bp of the first exons of thebSP-A1 orbSP-A2 gene linked to the human growth hormone (hGH) structural gene as a reporter were constructed. bSP-A1 andbSP-A2 genomic sequences were amplified from bSP-A1 andbSP-A2 genomic clones (13) by PCR with specific oligonucleotides containing sequences complementary to the 5′- and 3′-ends of the genomic regions to be amplified withHind III restriction sites near their 5′-ends and BamH I sites near their 3′-ends. PCR was accomplished withTaq polymerase (Boehringer Mannheim) and a DNA thermal cycler (Perkin-Elmer Cetus) with protocols suggested by the manufacturer. PCR fragments were digested withHind III orBamH I and subcloned into pACsk20GH, which contains the left 17% of the human adenovirus 5 genome and the promoterless hGH structural gene.

To generate recombinant adenoviruses, 293 cells, a permissive human embryonic kidney cell line, were cotransfected with the fusion genes and pJM17, which contains the entire adenovirus genome plus insertion of a 4.3-kb plasmid. pJM17 itself is too large to be packaged into viral particles. Infectious viral particles are formed on in vivo recombination of the plasmids to produce a recombinant viral genome of packageable size. Viral DNA was analyzed for the presence of the fusion genes by restriction endonuclease digestion and DNA sequencing. The number of infectious recombinant viruses were titered with 293 cells at least twice to ensure the accuracy of the titer.

Expression of SP-A fusion genes in transfected type II cells. Type II cells plated at a density of 5–9 × 106 cells/60-mm dish were maintained overnight in Waymouth MB 752/1 medium containing 10% fetal bovine serum. The cells were then washed twice with medium and incubated for 1 h with 1 × 106 recombinant viral particles, resulting in a multiplicity of infection of 0.1–0.2. In this manner, the same number of cells (1 × 106) was infected in each experiment. The medium was then aspirated and replaced with fresh medium in the absence or presence of DBcAMP (1 mM). Medium from transfected cells was collected every 24 h and assayed for hGH by RIA (Nichols Institute).

RESULTS

Developmental changes in bSP-A1 and bSP-A2 gene expression in the baboon fetal lung. In an initial study by McCormick and Mendelson (27) of the regulation ofhSP-A gene expression, it was observed that the hSP-A1 andSP-A2 genes are differentially regulated during development and by cAMP. To determine whether the twobSP-A genes are also differentially regulated as their human counterparts, RNase protection assays were used to analyze the expression of bSP-A1 and bSP-A2 mRNA transcripts at different developmental stages. Two antisense RNA probes to regions of the bSP-A genes that differ betweenbSP-A1 andbSP-A2 were generated by in vitro transcription of their respective DNAs (lane 1, top bands, in both Figs. 1 and 2). An 18S rRNA probe was simultaneously used as a standard for the normalization of loading. Radiolabeled probes were annealed with total RNA from the lung tissues of fetal baboons of 92, 125, 140, 160, and 175 days gestational age (term 184 days), digested with RNase, and fractionated by denaturing polyacrylamide gel electrophoresis. To evaluate the specificity of these probes, sense RNA fragments of either bSP-A1 or bSP-A2 were also transcribed from their respective DNAs and analyzed for their ability to protect the antisense RNA probes from RNase digestion. As expected, the bSP-A1 probe was protected from RNase digestion only by bSP-A1 RNA (Fig.1 A, lanes 3 and 4), whereas the bSP-A2 probe was protected from RNase digestion only by bSP-A2 RNA (Fig. 2 A, lanes 3 and 4). This confirmed that each probe was specific for its respective mRNA. The protected sense probes were somewhat longer than the protected bSP-A1 and bSP-A2 mRNAs in the tissue samples because the sense probes contained 5′ and 3′ extensions that hybridized to part of the 3′ and 5′ extensions of the antisense probes. The expression levels of both bSP-A1 and bSP-A2 were analyzed by scanning densitometry and were normalized to 18S rRNA (Figs.1 B and2 B). As can be seen, both bSP-A1 and bSP-A2 mRNA levels were barely detectable through 140 days gestational age but were markedly increased by day 175. At 160 days gestational age, bSP-A1 mRNA transcripts were increased to levels comparable to those onday 175, whereas bSP-A2 mRNA transcripts were much lower compared with those on day 175. Reproducible findings were obtained with RNA samples from an independent gestational series of baboon fetal lungs. Therefore, expression of the bSP-A2gene appears to be induced later during fetal development than that of the bSP-A1 gene.

Fig. 1.

Developmental changes in baboon surfactant protein A1 (bSP-A1) gene expression.A:32P-labeled antisense RNA probe specific for bSP-A1 gene was transcribed from +3,678 to +3,827 bp ofbSP-A1 genomic DNA (lane 1) (13). A32P-labeled 18S rRNA probe was generated by in vitro transcription of pTRI RNA 18S. Radiolabeled probes were annealed with either 2.5 μg of yeast RNA (lane 2), 0.1 μg of sense RNA probes for bSP-A1 and bSP-A2 (bSP-A1sense and bSP-A2sense, respectively) generated by in vitro transcription of the corresponding genomic sequences (lanes 3 and4, respectively), or total RNA isolated from lung tissues of 92 (d92)-, 125 (d125)-, 140 (d140)-, 160 (d160)-, and 175 (d175)-day gestational age fetal baboons (lanes 59, respectively). After RNase treatment and denaturing polyacrylamide gel electrophoresis, radiolabeled RNA fragments were detected by autoradiography. B: autoradiogram was analyzed by scanning densitometry. Shown are arbitrary units for bSP-A1 mRNA that were normalized to 18S rRNA (bSP-A1/18S).

Fig. 2.

Developmental changes in bSP-A2 gene expression. A:32P-labeled antisense RNA probe specific for bSP-A2 gene was transcribed from +3,644 to +3,833 bp ofbSP-A2 genomic DNA (lane 1) (13). A32P-labeled 18S rRNA probe was generated by in vitro transcription of pTRI RNA 18S. Radiolabeled probes were annealed with either 2.5 μg of yeast RNA (lane 2), 0.1 μg of bSP-A1sense and bSP-A2sense generated by in vitro transcription of the corresponding genomic sequences (lanes 3 and4, respectively), or total RNA isolated from lung tissues of 92-, 125-, 140-, 160-, and 175-day gestational age fetal baboons (lanes 59, respectively). After RNase treatment and denaturing polyacrylamide gel electrophoresis, radiolabeled RNA fragments were detected by autoradiography. B: autoradiogram was analyzed by scanning densitometry. Shown are arbitrary units for bSP-A2 mRNA that were normalized to 18S rRNA (bSP-A2/18S).

Effects of DBcAMP on levels of bSP-A1 and bSP-A2 mRNA transcripts in cultured baboon fetal lung tissues.Previously, McCormick and Mendelson (27) observed that the hSP-A2 gene is far more responsive to the inductive effects of cAMP than thehSP-A1 gene. To analyze the effects of cAMP on bSP-A1 andbSP-A2 gene expression, lung tissues from 125-day gestational age fetal baboons were cultured in the absence (control) and presence of 1 mM DBcAMP and analyzed for bSP-A1 and bSP-A2 mRNA transcripts by RNase protection. Total RNA was isolated from baboon fetal lung tissues after 1, 3, or 5 days in culture. The RNA was annealed with either the bSP-A1 or bSP-A2 probe, digested with RNase, and fractionated by denaturing polyacrylamide gel electrophoresis. As shown in Fig. 3, levels of expression of both the bSP-A1 andbSP-A2 genes were markedly induced by DBcAMP after 5 days in culture; however, in this experiment, the time course for this response differed in that thebSP-A1 gene was induced to relatively high expression levels by DBcAMP after 1 day in culture, whereas a major effect of DBcAMP to increasebSP-A2 gene expression was not evident until day 5 in culture. In three independent experiments with fetal lung tissues from 125-day gestational age fetal baboons, both thebSP-A1 andbSP-A2 genes were found to be comparably induced by DBcAMP after 5 days in culture. On the other hand, the time courses of induction of the twobSP-A genes in response to cAMP were not consistent in all experiments; in one of the three experiments,bSP-A2 expression was also markedly induced by DBcAMP as early as day 1 of culture.

Fig. 3.

Effects of dibutyryl cAMP (DBcAMP) on bSP-A1 and bSP-A2 mRNA levels in lung explants from a 125-day gestational age fetal baboon. Lung explants were maintained in organ culture in control medium or medium containing 1 mM DBcAMP. Total RNA was isolated from tissues after 1, 3, and 5 days in culture and analyzed for bSP-A1 (A) and bSP-A2 (B) mRNA levels by RNase protection assay as described in Figs. 1 and 2. After RNase treatment and denaturing polyacrylamide gel electrophoresis, radiolabeled RNA fragments were detected by autoradiography.

Sequence analysis of the 5′-flanking regions of the bSP-A1 and bSP-A2 genes. To identify potentialcis-acting elements that regulatebSP-A gene expression in type II cells and the inductive effect of cAMP, we analyzed ∼1.2 kb of DNA flanking the 5′-ends of the bSP-A1 andbSP-A2 genes (13). An alignment of the 5′-flanking sequences of the twobSP-A genes with the twohSP-A genes is shown in Fig.4. A putative nuclear receptor binding site [cAMP response element for the SP-A promoter (CRESP-A); TGACCT], previously found to be functionally required for cAMP induction of promoter activity of the rabbitSP-A (30) and thehSP-A2 (39) genes in transfected type II cells, was present in the 5′-flanking regions of bothbSP-A genes (from −246 to −239 bp in bSP-A1 and −248 to −241 bp in bSP-A2). A GT-box element (GGGGTGGG), found to be essential for basal and cAMP regulation of the hSP-A2 gene, was perfectly conserved in the bSP-A1 gene from −66 to −59 bp and was present as GGGGTGTG in the bSP-A2 gene from −67 to −60 bp. As can be seen in Fig. 4, the 5′-flanking sequence of thebSP-A1 gene is more similar to that of the hSP-A1 gene, whereas the 5′-flanking sequence of the bSP-A2 gene is more similar to that of the hSP-A2 gene. This is exemplified by deletions just upstream of the CRESP-A element in both thebSP-A2 andhSP-A2 5′-flanking sequences.

Fig. 4.

Alignment of nucleotide sequences of 5′-flanking regions of the 2bSP-A genes and the 2 humanSP-A(hSP-A) genes. Sequences ofbSP-A1 andbSP-A2 were determined in this study by analysis of the 2 bSP-A genomic clones previously isolated (13). Sequence ofhSP-A1 was previously reported (20). Sequence of hSP-A2 was determined in a previous study in this laboratory (McCormick and Mendelson, unpublished observations). Sequences were aligned with Genalign program in IG-Molecular Biology Software System. Gaps were inserted to maximize the number of matches among sequences and to create optimal alignment of multiple sequences. Vertical lines, identical bases between 2 genes. Nucleotides numbered +1, transcription initiation sites (arrow) as defined for hSP-A2 gene (24). Boxes enclose conserved TATA box, cAMP response element for SP-A promoter (CRESP-A), GT box, and thyroid transcription factor-1 (TTF-1) binding elements (TBEs) identified in this study.

Characterization of TTF-1 binding sites within the proximal 5′-flanking regions of the two bSP-A genes. It has been recently reported that four TTF-1 binding sites are located between −231 to −168 bp within the 5′-flanking region of the mouseSP-A gene. To localize TTF-1 binding sites in the proximal 5′-flanking regions of thebSP-A1 andbSP-A2 genes, DNase I footprinting assays were carried out with a bacterially expressed GST-TTFHD peptide. Bacterially expressed GST peptide was used as a control. As shown in Fig.5, two distinct protected regions were found to be present upstream of each gene; these are designated asfootprinted region 1 (FP1) and FP2 forbSP-A1 and FP2 and FP3 forbSP-A2. FP2 represents a homologous region upstream of the two bSP-Agenes; however, FP2 within the 5′-flanking region of thebSP-A1 gene is longer than itsbSP-A2 counterpart.

Fig. 5.

DNase I footprinting of bSP-A1 and bSP-A2 promoters with recombinant TTF-1. DNA probes corresponding to −253 to +40 bp (nos. at left) of 5′-flanking region and 1st exon ofbSP-A1 gene and −255 to +40 bp of 5′-flanking region and 1st exon ofbSP-A2 gene were end labeled and incubated without protein (lane 2), with glutathione S-transferase (GST;lanes 3 and7), or with increasing amounts (▴) of GST-TTF-1 cDNA fragment containing the entire homeodomain region (GST-TTF; 5, 10, and 15 μg; lanes 46, respectively) at room temperature for 30 min. Samples were treated with DNase I for 2 min, and reactions were stopped rapidly by ethanol precipitation. Products were then fractionated on 6% polyacrylamide-urea gels. Lanes 1, Maxam and Gilbert (25) chemical sequencing ladders (G+A) for the corresponding DNA fragments. FP1, FP2, and FP3,footprinted regions 13, respectively.

Sequence analysis of FP2 revealed that a conserved TTF-1 binding site (CTCAAG) was near its 5′-end. An oligonucleotide [TTF-1 binding element (TBE) 2a] containing this conserved TTF-1 binding site was synthesized and used as radiolabeled probe for EMSA (Fig.6). As can be seen, radiolabeled TBE2a formed a strong complex with GST-TTFHD but not with GST. Coincubation with a 100- to 1,000-fold excess of a nonradiolabeled TTF-1 binding oligonucleotide [oligo(C)] from the thyroglobulin gene promoter effectively abolished complex formation as did an excess of nonradiolabeled TBE2a itself. On the other hand, an oligonucleotide in which the conserved TTF-1 binding sequence (CTCAAG) in TBE2a was mutated to CTGTGC (TBE2am) failed to compete for binding with the radiolabeled TBE2a probe. Similarly, a nonspecific oligonucleotide (5′-AGAGTGGGTGACCTTAGCCA-3′) failed to affect complex formation. These findings indicate that the CTCAAG sequence represents a TTF-1 binding site present in both thebSP-A1 andbSP-A2 genes.

Fig. 6.

Electrophoretic mobility shift assay (EMSA) of TBE2a with recombinant TTF-1 polypeptide. Purified GST or GST-TTF (2 μg) was incubated with32P-labeled TBE2a (TBE2a*). Nonradiolabeled competitors were added as indicated; oligo(C) represents a consensus TTF-1 binding sequence from thyroglobulin gene promoter. CTCAAG sequence in TBE2a was changed to CTGTGC in TBE2am. NS, nonspecific oligonucleotide. ▴, Increasing concentrations of competitors at 100×, 500×, and 1,000× molar excess of radiolabeled probe. Nos. at right andleft, bp.

The DNase I footprinting analysis suggested the presence of another TTF-1 binding site within FP2. Based on a similarity to known TTF-1 binding sequences in other genes (9), examination of the 3′ portion of FP2 revealed that the CTCTAG sequence within thebSP-A1 gene and the CCTAAG sequence within the bSP-A2 gene could possibly serve as additional TTF-1 binding sites. As shown in Fig.7, radiolabeled oligonucleotides corresponding to the 3′ portions of the FP2 region (B1-TBE2b and B2-TBE2b) formed complexes with GST-TTFHD. Excess amounts of nonradiolabeled B1-TBE2b and B2-TBE2b effectively competed with the corresponding labeled probes for binding to the GST-TTFHD peptide. When CTCTAG in B1-TBE2b was mutated to the sequence of nucleotides in its corresponding position in the bSP-A2gene (CTCCAA) or when CCTAAG in B2-TBE2b was mutated to the sequence of nucleotides in its corresponding position in thebSP-A1 gene (CCTTGG), these two mutagenized oligonucleotides (B1-TBE2bm and B2-TBE2bm) could no longer compete with the radiolabeled wild-type probes for binding to the GST-TTFHD peptide. These findings suggest that these sequences represent additional TTF-1 binding sites within the FP2 regions of their corresponding genes. Interestingly, the spacing of the two TTF-1 binding sites within the FP2 regions are different. There are 14 nucleotides present between TBE2a and TBE2b of the bSP-A1 gene, whereas there are only 5 nucleotides present between TBE2a and TBE2b of thebSP-A2 gene; this explains why the FP2 upstream of the bSP-A1 gene is wider than that of the bSP-A2 gene. Using similar competition EMSA, we determined that the CTGGAG sequence in FP1 (named TBE1) and the TTGTAG sequence in FP3 (named TBE3) represent additional TBEs within the 5′-flanking regions of thebSP-A1 andbSP-A2 genes, respectively (data not shown).

Fig. 7.

TBE2b represents a 2nd TTF-1 binding site within FP2. Purified GST or GST-TTF (2 μg) was incubated with32P-labeled TBE2b ofbSP-A1 gene (B1-TBE2b*) orbSP-A2 gene (B2-TBE2b*). Nonradiolabeled competitors were added at 500× molar excess of radiolabeled probes (lanes 4,5, 9, and 10). CTCTAG sequence in B1-TBE2b was changed to CTCCAA in B1-TBE2bm. CCTAAG sequence in B2-TBE2b was changed to CCTTGG in B2-TBE2bm. DNA-protein complexes were separated from free probe by 5% nondenaturing polyacrylamide gel electrophoresis and were visualized by autoradiography.

To further confirm that the TTF-1 binding sites identified with the bacterially expressed GST-TTFHDpeptide were also able to bind native TTF-1 protein, alveolar type II cell nuclear extracts were used in EMSA analysis. As shown in Fig.8 A, type II cell nuclear extracts formed a strong complex with radiolabeled TBE2a oligonucleotide (lane 2). The binding of radiolabeled TBE2a was effectively competed by a 500× molar excess of nonradiolabeled FP1 but was unaffected by a nonradiolabeled oligonucleotide for the corresponding region of thebSP-A2 gene (B2-F1; Fig.8 A, lanes 3 and 4). This again indicates that FP1 is present only in the 5′-flanking region of the bSP-A1 gene. Both nonradiolabeled B1-FP2 and B2-FP2 effectively competed with the TBE2a probe for binding to type II cell nuclear extracts (Fig.8 A, lanes 5 and 6), indicating that both oligonucleotides contain TTF-1 binding sites. In Fig.8 A, lanes 7 and 8, nonradiolabeled FP3 effectively competed for binding, whereas an oligonucleotide for the corresponding region of thebSP-A1 gene (B1-F3) did not affect complex formation, confirming that FP3 is present only in the 5′-flanking region of the bSP-A2gene. As shown in Fig. 8 B, this protein-DNA complex was displaced when TTF-1 antiserum was included in the binding-reaction mixture, indicating that TTF-1 is present in the type II cell nuclear protein-DNA complex.

Fig. 8.

EMSA with fetal lung type II cell nuclear protein extract (NE).A: NE from human fetal lung type II cells (type II NE) was incubated with32P-labeled TBE2a*. Various nonradiolabeled competitors [oligonucleotides for corresponding regions (B2-F1, B1-FP2, B2-FP2, and B1-F3)] were added at 500× molar excess of radiolabeled probe as indicated. DNA-protein complexes were separated from free probe by 5% nondenaturing polyacrylamide gel electrophoresis and were visualized by autoradiography. B: NE from human fetal lung type II cells was incubated with32P-labeled TBE2a* in absence and presence of 1 μl of TTF-1 antiserum (αTTF-1) as indicated. As a negative control, antiserum was added to binding reactions in absence (−NE) and presence (+NE) of NE. DNA-protein complexes were separated from free probe on a 5% nondenaturing polyacrylamide gel and were visualized by autoradiography.

Functional localization of genomic sequences that mediate basal and cAMP induction of bSP-A1 and bSP-A2 promoter activity in type II cells. To define the genomic regions upstream of the bSP-A1 andbSP-A2 genes that are required for cAMP regulation of bSP-A gene expression in type II cells, bSP-A-hGHfusion genes composed of 50, 253 or 255, and 1,068 or 986 bp of DNA flanking the 5′-ends of thebSP-A1 orbSP-A2 gene and 40 bp from the first exon, which encodes the 5′-untranslated region, linked to thehGH structural gene as a reporter were constructed. These fusion genes were incorporated into the genome of the replication-defective human adenovirus 5 for highly efficient and reproducible DNA transfer by infection of rat fetal lung type II cells in primary culture. Because the same number of infectious viral particles containing each fusion gene was used in all experiments and the number of infectious particles were limiting relative to the number of cells (multiplicity of infection ∼ 0.2), the same number of type II cells was infected with each construct in each experiment. This resulted in highly reproducible and comparable data from one experiment to another. To analyze the effects of cAMP on fusion gene expression, infected type II cells were incubated in the absence and presence of DBcAMP (1 mM); bSP-A promoter activities were analyzed by radioimmunoassay of hGH protein secreted into the culture medium over each 24-h period. As shown in Fig. 9, expression of both 50-bp bSP-A-hGHfusion gene constructs was barely detectable and unaffected by cAMP treatment. By contrast, the fusion genes containing −253 bp ofbSP-A1 or −255 bp ofbSP-A2 5′-flanking DNA, respectively, were expressed at comparably elevated basal levels and were induced approximately sevenfold by DBcAMP. The expression levels of the −1,068-bp bSP-A1-hGH or −986-bp bSP-A2-hGH fusion gene were greatly reduced compared with those of the −253-bpbSP-A1-hGH or −255-bpbSP-A2-hGH fusion gene, suggesting the presence of upstream inhibitory elements; however, the induction by cAMP remained similar to that observed with the −253-bpbSP-A1-hGH or −255-bpbSP-A2-hGH fusion gene. Again, the basal and cAMP-induced expression levels of the −1,068-bpbSP-A1-hGH and −986-bpbSP-A2-hGH fusion genes were found to be comparable. These findings are consistent with the observation with RNase protection assays that expression of thebSP-A1 andbSP-A2 genes are highly induced by cAMP (Fig. 3). In studies using the transfected type II cells, there was no obvious difference in the time course of response of these fusion genes to cAMP treatment (data not shown). This is in contrast to the findings from RNase protection assays that the temporal effects of cAMP on expression of the bSP-A1 andbSP-A2 genes in cultured baboon fetal lung appear to be different (Fig. 3).

Fig. 9.

Expression of bSP-A-human growth hormone (hGH) fusion genes containing various amounts of 5′-flanking DNA frombSP-A1 andbSP-A2 genes in type II cells cultured in the absence and presence of DBcAMP. Rat fetal lung type II cells in monolayer culture were infected with recombinant adenoviruses containing +40 bp of exon I and 50, 253 or 255, or 1,068 or 986 bp of 5′-flanking DNA from bSP-A1 orbSP-A2 gene linked tohGH structural gene as a reporter. After infection, cells were incubated for up to 5 days in serum-free medium in absence and presence of 1 mM DBcAMP. Culture medium was harvested and replaced every 24 h; hGH concentrations shown are in medium collected on day 5 of culture. Values are means ± SE of data from 2 independent experiments, each conducted in triplicate. Negative subscript nos., bp of DNA flanking 5′-end of gene.

DISCUSSION

In the present study, the expression of bSP-A1 and bSP-A2 mRNA transcripts at different developmental stages was characterized with RNase protection assays. Consistent with previous findings for the twohSP-A genes (27), the two bSP-A genes also appear to be differentially regulated during development. Both bSP-A1 and bSP-A2 mRNA levels were found to be barely detectable through 140 days gestational age. At 160 days gestational age, bSP-A1 mRNA transcripts were increased to levels comparable to those onday 175, whereas bSP-A2 mRNA transcripts were considerably lower than those on day 175. Therefore, high levels of expression of thebSP-A2 gene occur later during development compared with those of thebSP-A1 gene. By Northern analysis, the levels of total SP-A mRNA transcripts on day 160 were found to be comparable to those onday 175 (35), suggesting that at 160 days of gestation, bSP-A1 mRNA comprises the dominantSP-A gene transcripts. This result is consistent with the previous finding (27) that, in lung tissue from a 28-wk-gestation neonate, hSP-A1 mRNA transcripts represented the predominant SP-A gene transcripts. From the findings of the present study, it is unclear as to whether there are subsequent changes in the level of onebSP-A gene transcript relative to the other at term or postnatally.

The mechanisms for the apparent differential regulation of the twobSP-A genes during development are unclear. Because both genes are likely to be expressed in the same cell types, they should be accessed by the same complement of transcription factors. Differential regulation of the two genes is possibly due to differences in the DNA elements that mediate their regulation. The 5′-flanking sequences of the twobSP-A genes were therefore sequenced for analysis of putative cis-acting elements based on a similarity to previously characterized transcription factor binding sites. CRESP-A and a GT box, two previously characterized elements important for mediating basal and cAMP-induced expression of rabbit (1, 30) and human (39, 40) SP-A promoter activity in type II cells, were found to be conserved in the 5′-flanking regions of bothbSP-A genes. By a combination of DNase I footprinting and EMSA, three TTF-1 binding sites were characterized in the 5′-flanking region of eachbSP-A gene. As is the case for the previously characterized TBEs within otherSP gene promoters, sequences of the TBEs within the two bSP-A gene 5′-flanking sequences were found to be highly degenerate. The core sequences for TTF-1 binding in the twobSP-A genes include motifs of CTCAAG, CTCTAG, CTGGAG, CCTAAG, and TTGTAG. Interestingly, the position and spacing of these TTF-1 binding sites within the 5′-flanking sequences of the two bSP-A genes also are different. Only TBE2a is conserved between the twobSP-A genes. Within FP2, the spacing between TBE2a and TBE2b is wider in thebSP-A1 gene than in thebSP-A2 gene; this explains why FP2 upstream from bSP-A1 is longer than that of bSP-A2. The TBE in FP1 (TBE1) is present only in the bSP-A1 gene, whereas that in FP3 (TBE3) is present only in thebSP-A2 gene. Interestingly, TBE3 is adjacent to the previously characterized GT box, whereas TBE1 is close to the CRESP-A element. Whether this positioning could potentially facilitate protein-protein interactions and thereby contribute to differential regulation of the two bSP-A genes remains to be determined.

In studies using cultured lung explants from 125-day gestational age fetal baboons, both the bSP-A1 andbSP-A2 genes were found to be markedly induced by DBcAMP. This is in contrast to the finding by McCormick and Mendelson (27) that the hSP-A2 gene is far more responsive to the inductive effects of cAMP than thehSP-A1 gene. Because the sequences of the bSP-A1 andbSP-A2 genes are more similar to each other than are those of the hSP-A1 andhSP-A2 genes (13), it is likely that during evolution, divergence of thehSP-A1 gene resulted in a decrease in its responsiveness to cAMP.

To characterize the genomic sequences that mediate basal and cAMP-induced expression of the bSP-A1and bSP-A2 genes in type II cells, rat fetal lung type II cells in primary monolayer culture were transfected with fusion genes containing various amounts of 5′-flanking sequences from the bSP-A1 andbSP-A2 genes linked to thehGH structural gene as a reporter. We found that 253-bp bSP-A1 and 255-bpbSP-A2 5′-flanking sequences were sufficient to direct relatively high levels of basal expression in primary cultures of type II cells cultured in the absence of cAMP; expression was stimulated approximately sevenfold by the addition of DBcAMP to the culture medium. Both basal and cAMP-induced expression levels of −253-bpbSP-A1-hGH and −255-bpbSP-A2-hGH fusion genes were found to be comparable. The time courses of induction of these fusion genes in response to cAMP also were similar (data not shown), suggesting either that the mechanisms for the apparent differential regulation of the two bSP-A genes during development observed with RNase protection assays of baboon fetal lung RNA are not active in this system or that the DNA elements responsible for the differential regulation of the twobSP-A genes are not present within the fusion gene constructs. The 253- and 255-bp 5′-flanking regions of bSP-A1 andbSP-A2, respectively, contain the previously characterized CRESP-Aelement and the GT box; both of these elements have previously been found to be required for cAMP induction of SP-A promoter activity in transfected type II cells (30, 39). Interestingly, expression levels of the bSP-A1- andbSP-A2-hGH fusion genes containing −1,068-bp bSP-A1 or −986-bp bSP-A2 5′-flanking sequences were reduced compared with the expression of fusion genes containing −253- or −255-bp 5′-flanking DNA. A similar finding was also obtained in deletional analysis of the 5′-flanking region of hSP-A2gene (39), suggesting the presence of putative inhibitory elements within the upstream regions and their conservation among species. The location of these inhibitory elements and their functional importance in contributing to the tissue-specific and developmental regulation ofSP-A gene expression are unknown at present. More detailed deletional mapping of theSP-A upstream sequences is necessary for localization of these putative transcriptional silencers.

Recently, Li et al. (23) reported that cAMP-induced expression of theSP-A gene is mediated by protein kinase A-induced phosphorylation of TTF-1, resulting in its increased DNA-binding and transcriptional activity. In type II cell transfection studies, we observed that mutation of each TBE within the bSP-A2 5′-flanking region characterized in the present study caused a reduction in basal and cAMP-induced bSP-A2 promoter; mutation of TBE2a had the most deleterious effect (23). Although it is clear that the integrity of these TBEs is required for the maximal cAMP induction of SP-A promoter activity (23), the findings of the present study suggest that the differences in position and spacing of the TBEs between thebSP-A1 andbSP-A2 genes do not affect responsiveness to cAMP. We suggest that the cAMP-induced increase in TTF-1 phosphorylation and DNA-binding activity may provide the primary mechanism whereby cAMP induces SP-Agene expression, whereas differential regulation of expression of thebSP-A1 andbSP-A2 genes during development may involve other mechanisms, such as a unique cooperative interaction between transcription factors in the context of gene-specific differences in chromatin structure.

Acknowledgments

We are grateful to Margaret Smith for expert help with cell and tissue culture.

Footnotes

  • Address for reprint requests: C. R. Mendelson, Dept. of Biochemistry, The Univ. of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd., Dallas, TX 75235-9038.

  • This research was supported by National Heart, Lung, and Blood Institute (NHLBI) Grant U10-HL-52647. The baboon tissues were provided by the Bronchopulmonary Dysplasia Resource Center (San Antonio, TX) that was funded by NHLBI Grant HL-52636.

  • J. Li was supported by a predoctoral fellowship from the Chilton Foundation, Dallas, TX.

  • The nucleotide sequences reported in this paper have been submitted to GenBank with accession numbers AF061967 for baboon surfactant protein A1, AF061968 for baboon surfactant protein A2, and AF061969 for human surfactant protein A2 gene 5′-flanking sequences.

  • The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

REFERENCES

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