SPECIAL TOPIC
Pre- and Postnatal Lung Development, Maturation, and Plasticity
Human surfactant protein B promoter in transgenic mice: temporal, spatial, and stimulus-responsive regulation

Marlene Strayer1, Rashmin C. Savani1, Linda W. Gonzales1, Aisha Zaman1, Zheng Cui1, Edina Veszelovszky1, Emily Wood1, Ye-Shih Ho2, and Philip L. Ballard1

1 Division of Neonatology, Department of Pediatrics, The Children's Hospital of Philadelphia, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104; and 2 Institute of Environmental Health Sciences, Wayne State University, Detroit, Michigan 482021


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Surfactant protein B (SP-B) is a developmentally and hormonally regulated lung protein that is required for normal surfactant function. We generated transgenic mice carrying the human SP-B promoter (-1,039/+431 bp) linked to chloramphenicol acetyltransferase (CAT). CAT activity was high in lung and immunoreactive protein localized to alveolar type II and bronchiolar epithelial cells. In addition, thyroid, trachea, and intestine demonstrated CAT activity, and each of these tissues also expressed low levels of SP-B mRNA. Developmental expression of CAT activity and SP-B mRNA in fetal lung were similar and both increased during explant culture. SP-B mRNA but not CAT activity decreased during culture of adult lung, and both were reduced by transforming growth factor (TGF)-beta 1. Treatment of adult mice with intratracheal bleomycin caused similar time-dependent decreases in lung SP-B mRNA and CAT activity. These findings indicate that the human SP-B promoter fragment directs tissue- and lung cell-specific transgene expression and contains cis-acting elements involved in regulated expression during development, fetal lung explant culture, and responsiveness to TGF-beta and bleomycin-induced lung injury.

transforming growth factor-beta ; lung explant culture; bleomycin lung injury; dexamethasone; lung development


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

SURFACTANT PROTEIN B (SP-B) is an ~8-kDa hydrophobic protein that is critical to the functioning of pulmonary surfactant, a phospholipid-rich, surface-active material that stabilizes lung alveoli. SP-B expression in human lung is regulated during fetal development and by hormones in vitro (3). Infants with inherited SP-B deficiency have respiratory distress at birth and eventually die without lung transplantation (36). In the mouse, ablation of the SP-B gene results in neonatal lethality at term birth (15).

The human SP-B gene is expressed at low levels as early as 12 wk gestation, with mRNA content increasing to ~50% of the adult level at 24 wk (30). In the mouse fetus, SP-B mRNA is detected at ~16 days gestation and increases manyfold by term (16). Production of mature SP-B protein is delayed relative to mRNA, paralleling development of lamellar bodies in alveolar type II cells and appearance of functional alveolar surfactant (8). This temporal pattern is consistent with a role for SP-B in lamellar body genesis and formation/stability of the alveolar surfactant film. Infants born prematurely often develop respiratory distress, in part secondarily to a developmental deficiency of mature SP-B.

In vivo, SP-B has been detected only in airway Clara cells and alveolar type II cells of the lung. We and others have previously studied function of the human SP-B gene promoter in vitro (12, 13, 51). Transient transfection experiments using different cell lines have mapped cell type specificity to the region of the proximal promoter (-112/-78 bp) that contains binding sites for the transcription factors thyroid transcription factor (TTF)-1 and hepatocyte nuclear factor (HNF)-3. An adenoviral transgene construct containing these human SP-B promoter sequences (-641/+319) also showed lung cell type specificity in vivo when administered intratracheally to mice (48).

SP-B gene expression is under both positive and negative regulation. Dexamethasone and cAMP agonists increase SP-B expression in cultured fetal lungs of several species, including human (3). Transforming growth factor (TGF)-beta , which is expressed early in normal lung development and is increased during inflammation and injury, inhibits SP-B expression in cultured human lung (9). 12-O-tetradecanoyl phorbol-13-acetate (TPA), a phorbol ester that activates protein kinase C, also downregulates transcription of SP-B in a human lung cell line and lung explants (43, 53). Kumar et al. (28, 29) localized both TPA and TGF-beta responsiveness to the TTF-1/HNF-3 binding sites within the proximal SP-B promoter. TNF-alpha also downregulates SP-B promoter activity, and recent evidence implicates TTF-1 in this response (10, 53). One or more of these inflammatory mediators may have a role in the downregulation of SP-B that occurs in various models of acute lung injury, including instillation of bleomycin or endotoxin and infection with adenovirus or Pneumocystis carinii (7, 32, 45, 57).

In this study we generated transgenic mice carrying the human SP-B promoter (-1,039/+431 bp) linked to chloramphenicol acetyltransferase (CAT) reporter gene. We hypothesized that this promoter fragment would direct developmentally regulated tissue- and cell-specific transgene expression and that expression would be modulated by TGF-beta . We examined fetal, newborn, and adult transgenic mice for expression from both the exogenous human (CAT) and endogenous mouse SP-B promoters.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Generation of transgenic mice. The SP-B-CAT plasmid construct (-1,039/+431CODelta n) used to generate transgenic mice has previously been described (51). It contains -1,039/+431 bp of the human SP-B gene, deleted for the ATG at +14/+16, in frame with bacterial CAT gene and simian virus 40 polyA sequences.

Transgenic C57Bl/6 × C3H hybrid mice (B6C3) were generated using standard procedures of microinjection (27). Founder mice were identified by Southern blot analysis of EcoRI-digested tail DNA using 32P-labeled CAT DNA as probe. Further generations of transgenics were identified using tail DNA dot blots. Founders were bred with B6C3 nontransgenic mice, and F1 and F2 generations were sibling mated. However, few viable litters were obtained from these matings. To maintain transgenic lines, F3 mice were outbred to CD-1 mice (Charles River, Wilmington, MA) for four to five generations followed by up to eight generations of inbreeding. Transgenic lines 66 and 79 were bred to homozygosity, but line 91 produced only heterozygotes. For fetal ontogeny studies, heterozygous transgenic fetuses were used for CAT analysis, and both transgenic and wild-type fetuses were used for SP-B mRNA data.

Mice were housed in the Animal Care Facility of the Children's Hospital of Philadelphia under standard conditions with free access to food and water. All animal experimental protocols were reviewed and approved by the Animal Use and Care Committee. In studies using different tissues, adult mice were killed by exposure to CO2, and tissues were removed and rapidly frozen. For studies involving intestine, mice were fasted 24 h, and residual intestinal contents were removed.

Bleomycin treatment of mice. Eight- to ten-week-old transgenic homozygous mice (line 66, 30-40 g) were anesthetized with ketamine:xylazine (16:8 mg/kg), and the trachea was visualized through a midline vertical incision in the neck. Using an insulin syringe, we injected either saline (50 µl) or 3 U/kg of bleomycin sulfate (Bristol Myers Squibb, Princeton, NJ) in 50 µl saline into the trachea. The incision was closed with surgical clips, and the animals were allowed to recover. Lungs from control and saline- and bleomycin-treated animals were harvested on days 2 and 4 after treatments. The right lung was separated from the trachea, snap-frozen in liquid nitrogen, and stored at -70°C until further processing. Frozen tissue was pulverized and processed for RNA extraction and CAT activity assays. The left lung was used for immunostaining as described below.

Explant culture. For fetal mouse lung explant studies, mice were mated and checked daily for vaginal plugs; presence of a plug was considered embryonic day 1. All explanted lungs were dissected from 16-day fetuses. Left and right lungs from the same fetus were used as a control and treated pair, alternating the left and right lung as control. Incisions were made in each of the lobes to allow for better penetration of medium components, and lungs were placed individually in the wells of a 24-well culture dish with 200 µl of the appropriate medium. Cultures were incubated on a rocker platform in an atmosphere of 5% CO2 in air. Medium was replaced every day and consisted of serum-free Waymouth medium alone or supplemented with dexamethasone (10 nM).

For adult lung explant culture, lungs were dissected with removal of large airways, pooled, and chopped into ~1-mm3 pieces with a McIlwain chopper as previously described (26). Explants were dispersed on 35-mm tissue culture dishes and incubated on a rocker platform in an atmosphere of 95% O2 and 5% CO2. Medium was replaced every day and consisted of serum-free Waymouth's MB 752/1 medium alone or supplemented with 30 ng/ml TGF-beta 1 (Sigma, St. Louis, MO). For [35S]methionine incorporation, explants were incubated for 1 h in met/cys-free medium and then cultured for 4 h with [35S]met/cys. Label incorporated into protein was determined as previously described (46).

CAT assay and Western blot. Frozen mouse tissues and lung explants were sonicated in water and centrifuged (14,000 × g for 10 min). Supernatants were assayed in duplicate for CAT enzyme activity by measuring the transfer of [3H]acetyl group from coenzyme A to chloramphenicol using phase extraction and scintillation counting as described previously (51). All CAT assay results are expressed as counts per minute (cpm) per minute incubation per 1 mg protein.

Western blot analysis for SP-B used Novex polyacrylamide gels (Invitrogen, Carlsbad, CA) under reducing conditions and a polyclonal rabbit antibody (PT3) raised against bovine SP-B8 with enhanced chemiluminescence detection (DuPont-NEN, Bedford, MA) as previously described (9). Protein concentrations were determined using Bio-Rad Protein Assay dye (Bio-Rad Laboratories, Richmond, CA).

RNA isolation and hybridization. RNA was extracted using TRIzol reagent according to manufacturer's directions (Life Technologies, Gaithersburg, MD), and serial dilutions (4, 2, and 1 µg) were dotted onto nitrocellulose membrane (Duralose, Stratagene, Cedar Creek, TX). Blots were hybridized under high stringency conditions with [alpha -32P]dCTP random-primed probes (Ready-To-Go DNA Labelling Beads, Amersham Pharmacia Biotech, Piscataway, NJ) for rat SP-B cDNA (19) and either human beta -actin cDNA (30) or end-labeled oligonucleotide for 28S rRNA (6) as previously described (30). Blots were exposed to DuPont Reflection film with intensifying screens, and autoradiograms were scanned and analyzed using a Hoeffer GS300 densitometer and GS370 software (Hoeffer Scientific Instruments, San Francisco, CA).

RT-PCR. cDNA was made using total RNA following the manufacturer's protocol (RNA PCR Core Kit, Perkin Elmer, Branchburg, NJ) and using oligo dT to prime the reaction. Primers for mouse SP-B were 5'-GTGCTTGATGTCTACCTGC-3' (sense, cDNA bp 678-696) and 5'-TGCCTGTC- TAGCCAGAAG-3' (antisense, cDNA bp 1,307-1,324) and produced a 647-bp amplicon from mRNA. PCR was done in a standard reaction using 2 mM MgCl2 and Amplitaq gold (Perkin Elmer). Reaction parameters were 95°C for 5 min, then 40 cycles of 95°C for 30 s, 52°C for 30 s, 72°C for 45 s, followed by 4 min at 72°C, then 4°C. Five microliters of the 50 µl reaction mix were analyzed on a 1.8% agarose gel and stained with ethidium bromide.

PCR primers for beta -actin were 5'-AAGAGAGGCATCCTCACCCT-3' (sense, cDNA bp 261-280) and 5'-TACATGGCTGGGGTGTTGAA-3' (antisense, cDNA bp 259-280) and produced a 218-bp amplicon from mRNA. PCR was done in a standard reaction using 2.5 mM MgCl2 and Amplitaq gold. Reaction parameters for beta -actin were 94°C for 5 min, 35 cycles of 94°C for 45 s, 52°C for 45 s, 72°C for 45 s, followed by 5 min at 72°C, then 4°C. Three microliters of the 50 µl reaction mix were analyzed on an 8% PAGE gel (Invitrogen) and stained with SYBR green (Molecular Probes, Eugene, OR).

For sequencing of PCR products, bands were eluted from a 6% polyacrylamide gel, ethanol precipitated, and amplified by PCR. PCR products were isolated by agarose gel electrophoresis and extracted using Qiaquick gel extraction kit (Qiagen, Valencia, CA). DNA sequencing was done by a core facility using big dye terminator chemistry (Applied Biosystems, Foster City, CA).

Immunofluorescence. Lungs were instilled with 1% paraformaldehyde for 3 h, rinsed well with PBS, instilled with 5% sucrose, and the next day frozen in cryo-embedding medium. Frozen sections were stained with polyclonal antibodies made against rat SP-B (Chemicon, Temecula, CA), CAT (5' right-arrow 3', Boulder, CO) or rat SP-A (52). CAT antibody was preadsorbed against cell lysate of NCI-H441 cells (human adenocarcinoma) to reduce background. Cy3-conjugated goat anti-rabbit IgG was used as secondary antibody at a dilution of 1:3,000. Fluorescence was viewed with epifluorescence at 510-560 nm on a Nikon TE300 inverted microscope with appropriate ultraviolet filters. Images were captured using a Hamamatsu digital camera using Metamorph software (Universal Imaging, West Chester, PA).

Statistics. Data for mRNA levels were normalized to beta -actin or 28S rRNA. All values are given as means ± SE for each group. Statistical analyses for group mean data were carried out using Student's t-test. The level of significance was P < 0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Tissue and cell distribution of transgene expression. Increased CAT activity, compared with wild-type animals, was detected in lungs from 8 of 13 founder mice. An initial survey of CAT activity in various tissues was performed with three of the positive founder animals. CAT activity was significantly increased (22 ± 3 cpm · min-1 · mg protein-1) in lung tissue, but not in seven other tissues, compared with wild-type tissues (all <2 cpm · min-1 · mg protein-1).

Distribution of CAT activity was reexamined after inbreeding, subsequent outbreeding to CD1 mice, and additional inbreeding to establish homozygous transgenic mice. We studied three lines of SP-B promoter transgenic mice: 66, 79, and 91. Transgene copy number, assessed by relative signal intensity on dot blot hybridization of tail DNA, was similar in lines 66 and 91, and 5- to 10-fold higher in line 79. Transgene activity in lung was high in lines 66 and 91 and near the wild-type level in line 79 (Table 1), consistent with findings in other studies demonstrating that copy number is not associated with the level of transgene expression (1, 2, 22).

                              
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Table 1.   CAT activity in mouse tissues

Results for CAT activity in 16 different tissues of wild-type and the three transgenic mouse lines are summarized in Table 1. CAT activity in tissues of wild-type mice ranged from undetectable to 11 cpm · min-1 · mg protein-1, with most tissues having levels <= 1. CAT activity of transgenic tissues was significantly higher than wild-type in lung (lines 66 and 91) and trachea (all three lines) and unexpectedly was also detected in thyroid and intestine. CAT activity in lung tissue of lines 66 and 91 was 465 and 1,352 cpm · min-1 · mg protein-1, respectively, with corresponding values for trachea of 570 and 182 cpm · min-1 · mg protein-1, respectively. CAT activity was not elevated in lung of line 79 compared with wild-type, but activity in trachea (193 cpm · min-1 · mg protein-1) was comparable to the other transgenic lines. Expression of transgene in mouse trachea is consistent with the high proportion of Clara cells in tracheal epithelium in this species (39). Thyroid CAT activity was manyfold greater than lung in line 79, comparable to lung activity in line 66 and substantially less in line 91. There were modest increases in CAT activity of both small intestine and colon of lines 66 and 91 compared with wild-type. Intestinal CAT activity did not appear to represent residual luminal bacteria, as CAT levels were low in intestinal contents assayed separately (data not shown).

We performed RT-PCR analysis for SP-B mRNA to examine endogenous mouse SP-B promoter activity in trachea, thyroid, and intestine. A 647-bp SP-B RT-PCR product was observed in these tissues but not in heart and kidney (Fig. 1A). DNA sequencing of gel-isolated product from thyroid confirmed its identity as SP-B (data not presented). Lung, trachea, and thyroid also produced a second, slightly smaller RT-PCR product that represents a 69-bp deleted form of SP-B mRNA as previously described by Chi et al. (14). This product was not observed from PCR of cloned rat SP-B cDNA (data not shown). PCR for beta -actin was run from the same RT reaction as a control for RNA integrity and experimental conditions (Fig. 1A). All reactions produced the expected 218-bp beta -actin RT-PCR product. We also performed dot blot hybridization with RNA prepared from various tissues. A weak hybridization signal for SP-B mRNA was found for trachea (~1% relative to lung) with lower but detectable signals for thyroid and intestine but not other tissues (not shown). In Western blot analysis of six different mouse tissues, only lung exhibited a ~8-kDa SP-B band (Fig. 1B), and precursor forms of SP-B were not detected in any tissues under the conditions used (not shown).


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Fig. 1.   Surfactant protein (SP)-B mRNA and protein in different mouse tissues. A: RT-PCR analysis for SP-B (top) and beta -actin mRNA (bottom) as a control was performed with RNA from mouse lung (1), thyroid (2), large intestine (3), proximal small intestine (4), distal small intestine (5), heart (6), kidney (7), trachea (8). 9, water blank; M, size markers. Input RNA for cDNA reactions was 0.1 µg for lung and thyroid and 1 µg for other tissues. All other parameters were the same for the different tissues as described in MATERIALS AND METHODS. B: Western blot analysis for SP-B in mouse tissues. Lanes contain 25 µg protein from liver (1), thyroid (2), small intestine (3), lung (4), heart (5), and trachea (6).

Figure 2 illustrates the cellular distribution of transgenic CAT and endogenous SP-B immunoreactivity in transgenic and wild-type adult lung tissue. The SP-B antibody used in these studies recognizes both pro- and mature forms of the protein. SP-B localized to discrete alveolar cells, in a pattern consistent with type II cells, and to most epithelial lining cells of bronchioles. There is a similar distribution of immunoreactive CAT in transgenic lung tissue (middle panel). These results are consistent with expression of CAT transgene in alveolar type II cells and in Clara cells, which is the major cell type of airways in the mouse.


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Fig. 2.   Immunofluorescent localization of chloramphenicol acetyltransferase (CAT) and SP-B within lung. Frozen sections from transgenic (line 66, left and center) and wild-type (right) adult mouse lung were stained with polyclonal antibodies to SP-B or CAT (as noted) and visualized with immunofluorescence. Arrows denote Clara cells of a small airway, and arrowheads indicate alveolar cells with the expected distribution of type II cells.

Developmental regulation. To examine expression of the human SP-B promoter during fetal development, we assayed CAT activity and SP-B mRNA content in transgenic mouse fetuses of 15-19 days gestation as well as in newborn mice on day 1 of life. The developmental patterns for CAT activity and SP-B mRNA were comparable with low or undetectable levels at days 15-16 and maximal values obtained on postnatal day 1 (Fig. 3). These findings suggest that the human SP-B promoter, driving CAT transgene expression, is activated during development with a pattern similar to that for the endogenous mouse SP-B promoter.


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Fig. 3.   Ontogeny of SP-B mRNA and CAT activity in lung. Lungs from heterozygous transgenic mice (lines 66 and 91) of different gestational ages were assayed for SP-B mRNA (dashed line) and for CAT activity (solid line). SP-B mRNA data are means ± SE of 3-12 pups at each time point (lungs from day 16 gestation were pooled before extraction) and normalized to beta -actin mRNA levels. For CAT activity, data are means ± SE of 3-30 pups at each time point. Results for lines 66 and 91 were very similar, and data were combined. All data (SP-B mRNA and CAT activity) are expressed as percentage of the level on postnatal day 1.

Expression in explant culture. We also examined endogenous and transgene promoter expression in explant culture using both fetal day 16 and adult tissue. We studied changes induced by culture itself as well as the effects of known positive and negative regulators of SP-B expression.

Fetal lung of various species undergoes precocious maturation during explant culture in the absence of serum or stimulatory hormones, in part related to increasing content of endogenous cAMP (5). We examined the effect of explant culture of fetal lung from 16-day gestation transgenic mice. Culture for 2 days mice, in the absence of serum or hormones, resulted in an approximately ninefold increase in SP-B mRNA content and an ~60-fold increase in CAT activity (Fig. 4). Because there is minimal cell proliferation during explant culture in serum-free medium (26), these changes primarily reflect increased gene expression per cell.


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Fig. 4.   SP-B mRNA and CAT activity during fetal lung explant culture and effect of dexamethasone. A: effect of explant culture. Gestation day 16 lungs from transgenic line 66 were either frozen before culture (day 0) or grown in explant culture for the indicated number of days in Waymouth medium alone. Data are the means ± SE from 6 fetuses. B: effect of dexamethasone. In separate experiments gestation day 16 lungs were cultured in presence or absence of dexamethasone (10 nM) for 2 days. Data for SP-B mRNA are normalized to beta -actin. Values are means ± SE for 5 fetuses (SP-B mRNA) and 11 fetuses (CAT) and are expressed as treated/control ratio for paired lungs.

In separate experiments, we examined the effects of dexamethasone, a known inducer of SP-B in mouse and human fetal lung. Explants prepared from transgenic fetal lung tissue on day 16 were cultured 2 days in the presence or absence of 10 nM dexamethasone. This treatment increased endogenous SP-B mRNA approximately sevenfold but did not affect the level of CAT activity (Fig. 4B). This finding is consistent with earlier transfection experiments in which the human SP-B promoter fragment -1,039/+431 was not glucocorticoid responsive (51).

Previous studies using type II cells isolated from adult rat lung have shown a rapid loss of SP gene expression in vitro (17). To examine activity of the human SP-B promoter under ex vivo conditions, we cultured transgenic adult mouse lung as explants in serum-free culture medium. SP-B mRNA levels decreased to 29% of preculture levels at 3-5 days (Table 2). This decrease was not due to loss of tissue viability, as beta -actin mRNA levels and rate of [35S]methionine incorporation into total protein did not decrease during culture. In contrast to SP-B mRNA, CAT activity did not decrease during culture (107% of preculture levels, Table 2).

                              
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Table 2.   Effects of explant culture and TGF-beta 1 treatment on adult mouse lung

TGF-beta is a negative regulator of SP-B in human type II cells, and responsiveness has been localized to the proximal promoter (-112/-78 bp) (9, 28). To examine responsiveness of the human SP-B promoter fragment in situ, explants of transgenic adult mouse lung were cultured with and without TGF-beta 1 (30 ng/ml). TGF-beta 1 treatment significantly reduced both SP-B mRNA and CAT activity compared with control. Viability as assessed by beta -actin mRNA levels and [35S]methionine incorporation was not affected by TGF-beta 1. These results are consistent with the proposed mechanism of TGF-beta action involving TTF-1/HNF-3-binding sites in the proximal region of the SP-B promoter (28).

We recently reported that SP-B gene expression is downregulated during lung injury secondary to intratracheal administration of bleomycin (45) and postulated that this is secondary to transcriptional downregulation by TGF-beta and/or other inflammatory mediators. To examine responsiveness of the human SP-B promoter to lung injury, homozygous transgenic mice (line 66) were treated with bleomycin (or saline as control) under conditions that reproducibly cause respiratory distress after 4 days. Lungs were examined for content of SP-B mRNA (Fig. 5A) and CAT activity (Fig. 5B) at 2 and 4 days after intratracheal instillation of bleomycin. There were similar, time-dependent decreases in endogenous SP-B mRNA and transgenic CAT activity in bleomycin-treated mice compared with control (untreated) and saline-treated animals.


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Fig. 5.   Effect of bleomycin lung injury. Adult, homozygous transgenic mice (line 66) were given intratracheal saline or bleomycin, and lungs were assayed for SP-B mRNA (A) and CAT activity (B) after 2 and 4 days. Data are means ± SE for 4 or 5 animals in each group. *P < 0.05 vs. control (no instillation).

The decrease in SP-B mRNA and CAT activity could reflect reduced promoter activity and/or loss of type II cells secondary to lung injury. We examined the latter possibility using SP-A immunostaining as a marker for type II cells. In rats, we have found that bleomycin injury reduces SP-B and SP-C expression but not SP-A (45). Similarly, bleomycin-injured mouse lungs contained SP-A-positive type II cells with a distribution and signal intensity qualitatively comparable to saline-treated lungs. SP-A immunoreactivity was also observed in macrophages and in air spaces of injured lungs. Staining for SP-B was decreased or undetectable in type II cells of bleomycin lungs compared with saline lungs (Fig. 6). These findings are consistent with negative regulation of SP-B promoter activity that involves interaction of endogenous inflammatory mediators with proximal promoter elements after lung injury.


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Fig. 6.   Immunostaining for SP-A and SP-B in lung tissue from saline- and bleomycin-treated mice. Sections were immunostained and photographed under identical conditions using antisera to SP-A (top panels), SP-B (middle panels), or nonimmune IgG (bottom panels). Arrowheads indicate presumptive type II cells, and arrows show presumptive macrophages. Type II cells are present in bleomycin-treated lung (SP-A staining) but have decreased or absent SP-B immunostaining.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

SP-B is required for production of active surfactant and for normal lung function. Expression of this gene in the lung is restricted to surfactant-producing type II cells and to Clara cells, where the function of SP-B is unknown. Studies of the human SP-B promoter in vitro have defined important DNA elements for both its activity and cell line specificity; however, there have been no investigations of the promoter in vivo. In this study we developed transgenic mice to investigate properties of the human SP-B promoter in vivo. We found that the -1,039/+431-bp fragment conferred both tissue- and cell-selective transgene expression as well as responsiveness to developmental and TGF-beta regulation.

We examined three different transgenic mouse lines expressing CAT activity. In line 79 there was loss of CAT activity in lung with successive generations; however, expression in both trachea and thyroid remained high. This may reflect site of transgene integration in this line. Whereas CAT expression in the lung occurred in both alveolar type II and bronchiolar Clara cells, tracheal expression was presumably restricted to Clara cells. The findings of differential CAT activity in lung and trachea of line 79 may indicate that regulation of SP-B transcription differs for tracheal vs. bronchiolar Clara cells.

Lines 66 and 91 demonstrated similar characteristics with respect to tissue specificity, expression level, developmental expression, and stimulus-responsive regulation of the transgene. The major difference between these lines was the inability to breed line 91 to homozygosity. We test crossed 32 progeny (males and females) from heterozygous matings and found no transgene homozygotes, suggesting that homozygosity in line 91 is embryonically lethal.

In addition to that in the lung, CAT activity was detected in the trachea, thyroid, and, at low levels, in intestine. Using RT-PCR and dot blot hybridization, we found low levels of SP-B transcripts (<= 1% of lung) in trachea, thyroid and intestine, but not in other tissues surveyed, providing the first demonstration of SP-B gene expression in these nonpulmonary tissues. Expression of CAT and SP-B mRNA in the trachea reflects the presence of Clara cells in this tissue (39), and pro-SP-B has been previously detected by immunohistochemistry in tracheal epithelial cells of fetal mice (56) and human fetuses and newborns (47). By Western blot analysis, mature SP-B was detected only in lung tissue. Our negative findings in the intestine do not necessarily contradict an earlier report of immunoreactive SP-B in intestinal mucosal scrapings because we examined the total intestine (18). Recently, SP-B was also detected in the porcine Eustachian tube (38), but we did not attempt to investigate this tissue in mice.

In agreement with our findings, Adams et al. (1) recently reported that transgenic mice containing the rabbit SP-B promoter (-730/+39) conferred type II- and Clara cell-specific expression of CAT reporter gene. Although the level of CAT activity in lung tissue was comparable to our data, CAT expression with the rabbit promoter was not detected in intestine and trachea, and thyroid was not examined. The different results for intestine and trachea may be related to promoter species, integration site, extent of 5'-flanking sequence, or relative sensitivities of the CAT assay.

We speculate that the SP-B promoter is active in the thyroid because of the presence of TTF-1, which is important for transcription of thyroid genes as well as the lung genes SP-A, SP-B, SP-C, and CC10 (11, 54). Previous studies of transgenic mice constructed with regions of these other promoters did not examine for transgene expression in the thyroid. Presumably, thyroid cells do not contain the enzymes that are required for pro-SP-B processing. This deficiency, in conjunction with relatively low mRNA content and rapid degradation of pro-SP-B, would account for undetectable SP-B protein under the conditions used. If expression of CAT in mouse thyroid reflects activation of human SP-B promoter by TTF-1, then it is likely that transgenes driven by other TTF-1-responsive promoters will also be expressed in thyroid as well as lung and trachea.

Developmental expression of CAT in mouse lung was nearly identical to that for endogenous SP-B mRNA. Considering plug day as day 1 of gestation, we detected low levels of CAT activity on day 15 and of SP-B mRNA on day 16, the earliest time points we examined. The findings for SP-B gene expression are consistent with previously reported data in the mouse (16). The CAT data imply that elements mediating responsiveness to developmental factors reside in the -1,039/+431-bp region of the human SP-B gene.

Explant culture of fetal lung has been used as a model for epithelial cell maturation. In agreement with other studies, we found that transgenic fetal mouse lungs from day 16 of gestation matured precociously when grown in explant culture, as reflected by a ninefold increase in endogenous SP-B message over 2 days. Similarly, CAT activity increased (60-fold) with 2 days of explant culture, consistent with culture-induced maturation mediated through elements present in the -1,039/+431-bp region. In studies with human fetal lung explants, culture-induced maturation of type II cells was associated with increasing content of endogenous cAMP (5). Moreover, an inhibitor of prostaglandin synthesis blocked the increases in both cAMP and SP-A mRNA, supporting a role for cAMP in the maturational process. This interpretation is consistent with the observed stimulatory effects of cAMP treatment on lung explants and isolated lung type II cells (25, 30, 37). It is possible, therefore, that the increases in both mouse SP-B mRNA and CAT activity during explant culture of fetal lung are related in part to stimulatory effects of endogenous cAMP and that cAMP-responsive elements reside within the promoter fragment used in these studies.

Analyses of the human SP-B gene promoter have demonstrated that in vitro activity is profoundly influenced by TTF-1 and HNF-3 interacting with elements in the proximal promoter (12, 13, 51). A distal enhancer region (-439/-331 bp) contains three additional TTF-1 binding sites as well as binding sites for retinoic acid receptors, and retinoic acid stimulates promoter activity in the presence of the TTF-1 binding sites (34). Furthermore, various cofactors (activator of thyroid and RA receptor, steroid coreceptor, steroid receptor coactivator, transcriptional intermediary factor-2, and BR22) have been shown to influence SP-B promoter activity in vitro (35, 55). It is conceivable that one or more of these factors could modulate SP-B promoter activity during in vivo development or during precocious maturation in explant culture.

We did not expect dexamethasone to increase CAT activity as was found for SP-B mRNA. In transfection studies with NCI-H441 cells, dexamethasone treatment had no effect on SP-B promoters from either the rabbit (-236/+39 bp) or human (-1,031/+439) (10, 51). Although glucocorticoid induction of human SP-B appears to be a primary response based on kinetics of induction and insensitivity to cycloheximide (4, 50), a glucocorticoid response element (GRE) has not been identified. Reichardt et al. (44) have reported that lung function is apparently normal in GR dimerization-deficient mice, raising the interesting possibility that glucocorticoids regulate SP-B (and other SPs) via non-GRE mechanisms.

Similar to the response observed on culture of isolated adult type II cells (17), SP-B mRNA levels decreased significantly during explant culture of adult mouse lung. By contrast, CAT activity was maintained during 5 days of explant culture. This difference likely does not reflect relative half-lives of SP-B mRNA vs. CAT protein, because CAT activity decreased with TGF-beta treatment. Lung tissue remained viable during explant culture, both with and without TGF-beta , as measured by beta -actin mRNA levels and [35S]methionine incorporation. The reasons for loss of differentiated gene expression in cultured adult lung are uncertain but could involve changes in hormonal milieu and tissue oxygen tension. Our data do not support the possibility that increased levels of endogenous active TGF-beta are involved in the process. If the loss of SP-B mRNA reflects decreased transcription, this response may involve elements not present in the human SP-B gene fragment.

The studies demonstrating decreased CAT activity after TGF-beta treatment of lung explants and with bleomycin lung injury in vivo provide new evidence that negative regulation of SP-B promoter activity is mediated via sites within the ~1-kb promoter fragment. In previous transfection studies with the human SP-B promoter, we localized responsiveness to both phorbol ester and TGF-beta to the proximal region (-112/-78 bp) that contains binding sites for TTF-1 and HNF-3. Treatment with either agent was associated with loss of TTF-1 DNA binding activity from the nucleus with a time course consistent with the decrease in SP-B mRNA (28, 29). Furthermore, a recent study with the rabbit SP-B promoter suggests that tumor necrosis factor (TNF)-alpha has a similar mode of action (10). Thus the downregulation of endogenous SP-B as well as CAT activity in transgenic animals injured by bleomycin could involve TGF-beta and/or TNF-alpha , since both inflammatory mediators are elevated after bleomycin treatment and are implicated in the pathobiology of the injury (21, 33, 41, 42).

Our studies identify a new genomic sequence to drive lung cell-specific transgene expression in vivo. Previously, we demonstrated that adenovirus containing a shorter fragment of the SP-B promoter (-641/+319) also directed transgene expression to mouse trachea and alveolar type II and bronchiolar Clara cells of lung (48). In that study, activity of the human SP-B promoter in lung was comparable to that found with an adenovirus construct using Rous sarcoma virus (RSV) promoter. The human SP-B promoter was also equivalent to RSV promoter and ~10-fold more active than thymidine kinase promoter in transfection studies (51). Collectively, these results indicate that the human SP-B promoter is relatively strong and thus should be useful for selective expression of various transgenes in lung cells.

Two findings are relevant to future use of the human SP-B promoter in transgenic animals. First, ectopic expression of transgene will likely occur in trachea, thyroid, and intestine. Depending on the properties of the particular transgene, this could potentially affect those organs or have an indirect effect on lung function. Second, in this study we have not systematically examined the percentage of type II and Clara cells that expressed transgene. Although the immunofluorescence studies suggested that a high proportion of both cell types expressed CAT, variation in the number of target cells expressing transgene has been reported for a variety of promoters including SP-C (22).

SP-A, SP-C, and Clara cell secretory protein (CCSP or CC10) promoters have all been used previously to direct lung epithelial cell transgene expression in mice. Transgene driven by ~378 bp or more of 5'-flanking sequence of the rabbit SP-A gene was expressed predominantly in lung, but activity was also detected in heart, thymus, and spleen (2). In the lung, transgene was expressed in both alveolar and bronchiolar epithelial cells and was developmentally regulated in a manner similar to endogenous SP-A. The SP-C promoter has been used extensively to express transgenes in the developing lung. To date, most transgenics have used a 3.7-kb human SP-C promoter fragment. Early studies demonstrated lung-specific activity of this promoter (23, 24), although expression in thymus and other tissues has been reported in one transgenic line (20), likely due to integration site (40). Endogenous SP-C is limited to alveolar type II epithelial cells, whereas the 3.7-kb SP-C promoter expresses transgene in both alveolar type II and bronchiolar epithelial cells. Bronchiolar expression has been recently mapped to the -1,960/-860 SP-C promoter region (22). Transgene activity from the 3.7-kb human SP-C promoter was detected in the lung on fetal day 11 and increased markedly by day 13-14, preceding the increase in endogenous SP-C (24). Both endogenous SP-C and the 3.7-kb SP-C-driven transgene responded positively to dexamethasone in explant cultures of 13-day fetal lungs, but the responsive region has not been characterized. The CC10 promoter has been shown to direct transgene expression selectively to pulmonary Clara cells. In developmental studies, expression from the rabbit CC10 promoter occurred 2 days earlier than for the endogenous mouse promoter (31, 49). Thus lung specific promoters are available for two cell types, allowing transgene expression targeted to one or both cell types. Developmental expression and hormonal responsiveness with these promoters, relative to the endogenous gene, are variable and may reflect the fragment used and/or species differences.

The present study demonstrates that the first kilobase of the human SP-B promoter is sufficient to direct appropriate developmental expression that is restricted to type II and Clara cells in adult mouse lung. Transgene expression also occurred in thyroid and intestine, and these tissues were shown to also express endogenous SP-B mRNA but not mature protein. Thus this fragment of the human SP-B promoter also imparts appropriate tissue specificity across species. The possible function, if any, of SP-B gene expression in nonpulmonary tissues is not currently known but does not appear to involve production of mature protein. The new findings regarding SP-B gene expression in thyroid and downregulation of the hSP-B promoter in bleomycin-injured lungs support an important role for TTF-1 in regulating SP-B gene expression in vivo.


    ACKNOWLEDGEMENTS

We thank Yue Ning, Ankur Gupta, and Sree Angampalli for technical assistance, and Sandy Mosiniak for editorial assistance.


    FOOTNOTES

This work supported by National Heart, Lung, and Blood Institute Grants HL-19,737 (M. Strayer, P. L. Ballard), HL-56401 (L. W. Gonzales, P. L. Ballard), HL-62472 (R. C. Savani), and HL-56421 (Y.-S. Ho) and The Gisela and Dennis Alter Endowed Chair in Pediatrics (L. W. Gonzales, P. L. Ballard).

Address for reprint requests and other correspondence: P. L. Ballard, Dept. of Pediatrics, Children's Hospital of Philadelphia, 416 Abramson Research Center, 3516 Civic Center Blvd., Philadelphia PA 19104-4318 (E-mail: ballardp{at}emailchop.edu).

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

10.1152/ajplung.00188.2001

Received 30 May 2001; accepted in final form 25 June 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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