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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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)-
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-
and bleomycin-induced lung injury.
transforming growth factor-; lung explant culture; bleomycin
lung injury; dexamethasone; lung development
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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)-, 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-
responsiveness to the TTF-1/HNF-3
binding sites within the proximal SP-B promoter. TNF-
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-
. 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Generation of transgenic mice.
The SP-B-CAT plasmid construct (1,039/+431CO
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.
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-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 [-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
-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 forImmunofluorescence.
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' 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 -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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 · min1 · 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).
|
|
|
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.
|
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.
|
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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-
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- treatment. Lung tissue
remained viable during explant culture, both with and without TGF-
,
as measured by
-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-
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-
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-
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)-
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-
and/or
TNF-
, 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Adams, CC,
Alum MN,
Starcher BC,
and
Boggaram V.
Cell-specific and developmental regulation of rabbit surfactant protein B promoter in transgenic mice.
Am J Physiol Lung Cell Mol Physiol
280:
L724-L731,
2001
2.
Alcorn, JL,
Hammer RE,
Graves KR,
Smith ME,
Maika SD,
Michael LF,
Gao E,
Wang Y,
and
Mendelson CR.
Analysis of genomic regions involved in regulation of the rabbit surfactant protein A gene in transgenic mice.
Am J Physiol Lung Cell Mol Physiol
277:
L349-L361,
1999
3.
Ballard, PL.
Hormonal regulation of pulmonary surfactant.
Endocr Rev
10:
165-181,
1989[ISI][Medline].
4.
Ballard, PL,
Ertsey R,
Gonzales LW,
and
Gonzales J.
Transcriptional regulation of human pulmonary surfactant proteins SP-B and SP-C by glucocorticoids.
Am J Respir Cell Mol Biol
14:
599-607,
1996[Abstract].
5.
Ballard, PL,
Gonzales LW,
Williams MC,
Roberts JM,
and
Jacobs MM.
Differentiation of type II cells during explant culture of human fetal lung is accelerated by endogenous prostanoids and adenosine 3',5'-monophosphate.
Endocrinology
128:
2916-2924,
1991[Abstract].
6.
Barbu, V,
and
Dautry F.
Northern blot normalization with a 28S rRNA oligonucleotide probe.
Nucleic Acids Res
17:
7115,
1989[ISI][Medline].
7.
Beers, MF,
Atochina EN,
Preston AM,
and
Beck JM.
Inhibition of lung surfactant protein B expression during Pneumocystis carinii pneumonia in mice.
J Lab Clin Med
133:
423-433,
1999[ISI][Medline].
8.
Beers, MF,
Shuman H,
Liley HG,
Floros J,
Gonzales LW,
Yue N,
and
Ballard PL.
Surfactant protein B in human fetal lung: developmental and glucocorticoid regulation.
Pediatr Res
38:
668-675,
1995[Abstract].
9.
Beers, MF,
Solarin KO,
Guttentag SH,
Rosenbloom J,
Kormilli A,
Gonzales LW,
and
Ballard PL.
Transforming growth factor-1 inhibits surfactant component expression and epithelial cell maturation in cultured human fetal lung.
Am J Physiol Lung Cell Mol Physiol
275:
L950-L960,
1998
10.
Berhane, K,
Margana RK,
and
Boggaram V.
Characterization of rabbit SP-B promoter region responsive to downregulation by tumor necrosis factor-.
Am J Physiol Lung Cell Mol Physiol
279:
L806-L814,
2000
11.
Bingle, CD.
Thyroid transcription factor-1.
Int J Biochem Cell Biol
29:
1471-1473,
1997[ISI][Medline].
12.
Bohinski, RJ,
Di Lauro R,
and
Whitsett JA.
The lung-specific surfactant protein B gene promoter is a target for thyroid transcription factor 1 and hepatocyte nuclear factor 3, indicating common factors for organ-specific gene expression along the foregut axis.
Mol Cell Biol
14:
5671-5681,
1994[Abstract].
13.
Bohinski, RJ,
Huffman JA,
Whitsett JA,
and
Lattier DL.
Cis-active elements controlling lung cell-specific expression of human pulmonary surfactant protein B gene.
J Biol Chem
268:
11160-11166,
1993
14.
Chi, X,
Garnier G,
Hawgood S,
and
Colten HR.
Identification of a novel alternatively spliced mRNA of murine pulmonary surfactant protein B.
Am J Respir Cell Mol Biol
19:
107-113,
1998
15.
Clark, JC,
Wert SE,
Bachurski CJ,
Stahlman MT,
Stripp BR,
Weaver TE,
and
Whitsett JA.
Targeted disruption of the surfactant protein B gene disrupts surfactant homeostasis, causing respiratory failure in newborn mice.
Proc Natl Acad Sci USA
92:
7794-7798,
1995[Abstract].
16.
D'Amore-Bruno, MA,
Wikenheiser KA,
Carter JE,
Clark JC,
and
Whitsett JA.
Sequence, ontogeny, and cellular localization of murine surfactant protein B mRNA.
Am J Physiol Lung Cell Mol Physiol
262:
L40-L47,
1992
17.
Dobbs, LG.
Isolation and culture of alveolar type II cells.
Am J Physiol Lung Cell Mol Physiol
258:
L134-L147,
1990
18.
Eliakim, R,
DeSchryver-Kecskemeti K,
Nogee L,
Stenson WF,
and
Alpers DH.
Isolation and characterization of a small intestinal surfactant-like particle containing alkaline phosphatase and other digestive enzymes.
J Biol Chem
264:
20614-20619,
1989
19.
Emrie, PA,
Shannon JM,
Mason RJ,
and
Fisher JH.
cDNA and deduced amino acid sequence for the rat hydrophobic pulmonary surfactant-associated protein, SP-B.
Biochim Biophys Acta
994:
215-221,
1989[ISI][Medline].
20.
Enelow, RI,
Stoler MH,
Srikiatkhachorn A,
Kerlakian C,
Agersborg S,
Whitsett JA,
and
Braciale TJ.
A lung-specific neo-antigen elicits specific CD8+ T cell tolerance with preserved CD4+ T cell reactivity. Implications for immune-mediated lung disease.
J Clin Invest
98:
914-922,
1996
21.
Giri, SN,
Hyde DM,
and
Hollinger MA.
Effect of antibody to transforming growth factor beta on bleomycin induced accumulation of lung collagen in mice.
Thorax
48:
959-966,
1993[Abstract].
22.
Glasser, SW,
Burhans MS,
Eszterhas SK,
Bruno MD,
and
Korfhagen TR.
Human SP-C gene sequences that confer lung epithelium-specific expression in transgenic mice.
Am J Physiol Lung Cell Mol Physiol
278:
L933-L945,
2000
23.
Glasser, SW,
Korfhagen TR,
Bruno MD,
Dey C,
and
Whitsett JA.
Structure and expression of the pulmonary surfactant protein SP-C gene in the mouse.
J Biol Chem
265:
21986-21991,
1990
24.
Glasser, SW,
Korfhagen TR,
Wert SE,
Bruno MD,
McWilliams KM,
Vorbroker DK,
and
Whitsett JA.
Genetic element from human surfactant protein SP-C gene confers bronchiolar-alveolar cell specificity in transgenic mice.
Am J Physiol Lung Cell Mol Physiol
261:
L349-L356,
1991
25.
Gonzales, LW,
Angampalli S,
Guttentag SH,
Beers MF,
Solarin KO,
Feinstein SI,
Matlapudi A,
and
Ballard PL.
Maintenance of differentiated function of the surfactant system in human fetal lung type II epithelial cells on plastic.
Pediatr Path Molec Med
20:
387-412,
2001[ISI].
26.
Gonzales, LW,
Ballard PL,
Ertsey R,
and
Williams MC.
Glucocorticoids and thyroid hormones stimulate biochemical and morphological differentiation of human fetal lung in organ culture.
J Clin Endocrinol Metab
62:
678-691,
1986[Abstract].
27.
Hogan, B,
Beddington R,
Costantini F,
and
Lacy E.
Manipulating the Mouse Embryo: A Laboratory Manual (2nd ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 1994.
28.
Kumar, AS,
Gonzales LW,
and
Ballard PL.
Transforming growth factor-1 regulation of surfactant protein B gene expression is mediated by protein kinase-dependent intracellular translocation of thyroid transcription factor-1 and hepatocyte nuclear factor 3.
Biochim Biophys Acta
1492:
45-55,
2000[ISI][Medline].
29.
Kumar, AS,
Venkatesh VC,
Planer BC,
Feinstein SI,
and
Ballard PL.
Phorbol ester down-regulation of lung surfactant protein B gene expression by cytoplasmic trapping of thyroid transcription factor-1 and hepatocyte nuclear factor 3.
J Biol Chem
272:
20764-20773,
1997
30.
Liley, HG,
White RT,
Warr RG,
Benson BJ,
Hawgood S,
and
Ballard PL.
Regulation of messenger RNAs for the hydrophobic surfactant proteins in human lung.
J Clin Invest
83:
1191-1197,
1989[ISI][Medline].
31.
Margraf, LR,
Finegold MJ,
Stanley LA,
Major A,
Hawkins HK,
and
DeMayo FJ.
Cloning and tissue-specific expression of the cDNA for the mouse Clara cell 10kD protein: comparison of endogenous expression to rabbit uteroglobin promoter-driven transgene expression.
Am J Respir Cell Mol Biol
9:
231-238,
1993[ISI][Medline].
32.
Mora, R,
Arold S,
Marzan Y,
Suki B,
and
Ingenito EP.
Determinants of surfactant function in acute lung injury and early recovery.
Am J Physiol Lung Cell Mol Physiol
279:
L342-L349,
2000
33.
Munger, JS,
Huang X,
Kawakatsu H,
Griffiths MJD,
Dalton SL,
Wu J,
Pittet JF,
Kaminski N,
Garat C,
Matthay MA,
Rifkin DB,
and
Sheppard D.
The integrin v
6 binds and activates latent TGF
1: a mechanism for regulating pulmonary inflammation and fibrosis.
Cell
96:
319-328,
1999[ISI][Medline].
34.
Naltner, A,
Ghaffari M,
Whitsett JA,
and
Yan C.
Retinoic acid stimulation of the human surfactant protein B promoter is thyroid transcription factor 1 site-dependent.
J Biol Chem
275:
56-62,
2000
35.
Naltner, A,
Wert S,
Whitsett JA,
and
Yan C.
Temporal/spatial expression of nuclear receptor coactivators in the mouse lung.
Am J Physiol Lung Cell Mol Physiol
279:
L1066-L1074,
2000
36.
Nogee, LM,
de Mello DE,
Dehner LP,
and
Colten HR.
Brief report: deficiency of pulmonary surfactant protein B in congenital alveolar proteinosis.
N Engl J Med
328:
406-410,
1993
37.
Odom, MJ,
Snyder JM,
and
Mendelson CR.
Adenosine 3',5'-monophosphate analogs and beta-adrenergic agonists induce the synthesis of the major surfactant apoprotein in human fetal lung in vitro.
Endocrinology
121:
1155-1163,
1987[Abstract].
38.
Paananen, R,
Glumoff V,
Sormunen R,
Voorhout W,
and
Hallman M.
Expression and localization of lung surfactant protein B in Eustachian tube epithelium.
Am J Physiol Lung Cell Mol Physiol
280:
L214-L220,
2001
39.
Pack, RJ,
Al-Ugaily LH,
Morris G,
and
Widdicombe JG.
The distribution and structure of cells in the tracheobronchial epithelium of the mouse.
Cell Tissue Res
208:
65-84,
1980[ISI][Medline].
40.
Palmiter, RD,
and
Brinster RL.
Germ-line transformation of mice.
Annu Rev Genet
20:
465-499,
1986[ISI][Medline].
41.
Phan, SH,
and
Kunkel SL.
Lung cytokine production in bleomycin-induced pulmonary fibrosis.
Exp Lung Res
18:
29-43,
1992[ISI][Medline].
42.
Piguet, PF,
Collart MA,
Grau GE,
Kapanci Y,
and
Vassalli P.
Tumor necrosis factor/cachectin plays a key role in bleomycin-induced pneumopathy and fibrosis.
J Exp Med
170:
655-663,
1989[Abstract].
43.
Planer, BC,
Ning Y,
Kumar SA,
and
Ballard PL.
Transcriptional regulation of surfactant proteins SP-A and SP-B by phorbol ester.
Biochim Biophys Acta
1353:
171-179,
1997[ISI][Medline].
44.
Reichardt, HM,
Kaestner KH,
Tuckermann J,
Kretz O,
Wessely O,
Bock R,
Gass P,
Schmid W,
Herrlich P,
Angel P,
and
Schutz G.
DNA binding of the glucocorticoid receptor is not essential for survival.
Cell
93:
531-541,
1998[ISI][Medline].
45.
Savani, RC,
Godinez RI,
Godinez MH,
Wentz E,
Zaman A,
Cui Z,
Pooler PM,
Guttentag SH,
Beers MF,
Gonzales LK,
and
Ballard PL.
Respiratory distress after intratracheal bleomycin: selective deficiency of surfactant proteins B and C.
Am J Physiol Lung Cell Mol Physiol
281:
L685-L696,
2001
46.
Solarin, KO,
Ballard PL,
Guttentag SH,
Lomax CA,
and
Beers MF.
Expression and glucocorticoid regulation of surfactant protein C in human fetal lung.
Pediatr Res
42:
356-364,
1997[Abstract].
47.
Stahlman, MT,
Gray ME,
and
Whitsett JA.
The ontogeny and distribution of surfactant protein B in human fetuses and newborns.
J Histochem Cytochem
40:
1471-1480,
1992
48.
Strayer, MS,
Guttentag SH,
and
Ballard PL.
Targeting type II and clara cells for adenovirus-mediated gene transfer using the surfactant protein B promoter.
Am J Respir Cell Mol Biol
18:
1-11,
1998
49.
Stripp, BR,
Sawaya PL,
Luse DS,
Wikenheiser KA,
Wert SE,
Huffman JA,
Lattier DL,
Singh G,
Katyal SL,
and
Whitsett JA.
Cis-acting elements that confer lung epithelial cell expression of the CC10 gene.
J Biol Chem
267:
14703-14712,
1992
50.
Venkatesh, VC,
Ballard PL,
Ertsey R,
and
Iannuzzi DM.
Glucocorticoid regulation of the genes for pulmonary surfactant proteins SP-B and SP-C.
Am J Respir Cell Mol Biol
8:
222-228,
1993[ISI][Medline].
51.
Venkatesh, VC,
Planer BC,
Schwartz M,
Vanderbilt JN,
White RT,
and
Ballard PL.
Characterization of the promoter of human pulmonary surfactant protein B gene.
Am J Physiol Lung Cell Mol Physiol
268:
L674-L682,
1995
52.
Wali, A,
Beers MF,
Dodia C,
Feinstein SI,
and
Fisher AB.
ATP and adenosine 3',5'-cyclic monophosphate stimulate the synthesis of surfactant protein A in rat lung.
Am J Physiol Lung Cell Mol Physiol
264:
L431-L437,
1993
53.
Whitsett, JA,
Clark JC,
Wispe JR,
and
Pryhuber GS.
Effects of TNF- and phorbol ester on human surfactant protein and MnSOD gene transcription in vitro.
Am J Physiol Lung Cell Mol Physiol
262:
L688-L693,
1992
54.
Whitsett, JA,
and
Glasser SW.
Regulation of surfactant protein gene transcription.
Biochim Biophys Acta
1408:
303-311,
1998[ISI][Medline].
55.
Yang, Y-S,
Yang M-CW,
Wang B,
and
Weissler JC.
BR22, a novel protein, interacts with thyroid transcription factor-1 and activates the human surfactant protein B promoter.
Am J Respir Cell Mol Biol
24:
30-37,
2001
56.
Zhou, L,
Lim L,
Costa RH,
and
Whitsett JA.
Thyroid transcription factor-1, hepatocyte nuclear factor-3 beta, surfactant protein B, C, and Clara cell secretory protein in developing mouse lung.
J Histochem Cytochem
44:
1183-1193,
1996
57.
Zsengeller, ZK,
Wert SE,
Bachurski CJ,
Kirwin KL,
Trapnell BC,
and
Whitsett JA.
Recombinant adenoviral vector disrupts surfactant homeostasis in mouse lung.
Hum Gene Ther
8:
1331-1344,
1997[ISI][Medline].