1 Pulmonary and Critical Care
Division, Transforming growth factor-
transforming growth factor- PULMONARY SURFACTANT is a surface-active mixture of
phospholipid and protein secreted by the alveolar type II cell that
reduces surface tension at the air-liquid interface and allows for
maintenance of alveolar stability at low lung volumes (32). The
predominant lipid synthesized is dipalmitoylphosphatidylcholine, which
accounts for much of the surface activity. The biosynthetic pathway for dipalmitoylphosphatidylcholine is complex, but it appears to involve both de novo synthesis as well as remodeling of previously synthesized lipid components. A key enzyme involved in the synthesis of new phospholipid is fatty acid synthetase (FAS), a rate-limiting and hormonally regulated enzyme that catalyzes conversion of malonyl-CoA to
fatty acid (31). In addition to phospholipids, surfactant also contains
several unique proteins designated surfactant protein (SP) A, SP-B,
SP-C, and SP-D (reviewed in Refs. 3, 32). SP-A regulates lipid
secretion and uptake in vitro and contributes to local host defense.
Two other, smaller lipophilic proteins, SP-B and SP-C, each confer
properties of rapid surface adsorption to synthetic phospholipid
mixtures. In humans, the expression of SP-C is limited to the alveolar
type II cell, whereas SP-A and SP-B expression have been shown to be
more promiscuous, having also been documented in other airway epithelia
(27). SP-D, like SP-A, is also important for local lung host defense
but has not been shown to play a role in surfactant homeostasis or in
the modulation of its biophysical function.
A developmental deficiency of surfactant is the major cause of
respiratory distress syndrome (RDS) in premature infants (1), and there
is an emerging body of evidence that dysfunction of the surfactant
system (either a relative deficiency or inactivation of normal
biophysical properties) also contributes to the pathogenesis of both
congenital and acquired lung disease (45). Inherited SP-B deficiency as
well as conditions such as acute RDS and Pneumocystis carinii pneumonia is associated with alterations in
surfactant lipids, proteins, and biophysical function (10, 14, 17, 45).
RDS can progress to chronic lung disease (CLD). Although many factors
contribute to the development of CLD, pulmonary inflammation may have a
central role in the pathogenesis (2, 15, 19, 20, 47). Recently, an
association between an early postnatal rise in bronchoalveolar lavage
levels of the multifunctional cytokine transforming growth factor
(TGF)- TGF- Studies (16, 33) using fetal mouse lung have shown that TGF- Overexpression of TGF- The human fetal lung explant model has been used to study the
expression and processing of SP-A, -B, and -C in early gestation (3, 4,
23, 34, 35, 40, 42-44). Although various factors have been shown
to influence expression of these SPs in this model, the effect of
TGF- In this study, we investigated the effects of exogenously administered
TGF- Materials
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
1 (TGF-
1) is a
multifunctional cytokine shown to play a critical role in organ
morphogenesis, development, growth regulation, cellular
differentiation, gene expression, and tissue remodeling after injury.
We examined the effect of exogenously administered TGF-
1 on the
expression of surfactant proteins (SPs) and lipids, fatty acid
synthetase, and ultrastructural morphology in human fetal lung cultured
for 5 days with and without dexamethasone (10 nM). Expression of the type II cell-specific marker surfactant proprotein C (proSP-C), studied
by [35S]Met
incorporation and immunoprecipitation, increased sevenfold with
dexamethasone treatment. TGF-
1 (0.1-100 ng/ml) in the presence of dexamethasone inhibited 21-kDa proSP-C expression in a
dose-dependent manner (maximal inhibition 31% of control level at 100 ng/ml). There was no change in
[35S]Met incorporation
into total protein in any of the treatment groups vs. the control
group. In immunoblotting experiments, TGF-
1 blocked culture-induced
accumulation of SP-A and SP-B. Under the same conditions, TGF-
1
reduced mRNA content for SP-A, SP-B, and SP-C to 20, 38, and 41%,
respectively, of matched control groups but did not affect levels of
-actin mRNA. SP transcription rates after 24 h of exposure to
TGF-
1 were reduced to a similar extent (20-50% of control
level). In both control and dexamethasone-treated explants, TGF-
1
(10 ng/ml) also decreased fatty acid synthetase mRNA, protein, and
enzyme activity and the rate of
[3H]choline
incorporation into phosphatidylcholine. By electron microscopy,
well-differentiated type II cells lining potential air spaces were
present in explants cultured with dexamethasone, whereas exposure to
TGF-
1 with or without dexamethasone resulted in epithelial cells
lacking lamellar bodies. We conclude that exogenous TGF-
1 disrupts
culture-induced maturation of fetal lung epithelial cells and inhibits
expression of surfactant components through effects on gene
transcription.
1; human fetal lung explants; surfactant proteins; fatty acid synthetase; dexamethasone
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
1 and the development of CLD has been reported (20). TGF-
1
has also been associated with the increased expression of extracellular
matrix genes observed in biopsy specimens from adult humans with
idiopathic pulmonary fibrosis (6) and implicated as a mediator of the
type II cell proliferation seen in the bleomycin-treated rat model of
lung injury and repair (18).
1 is a cytokine notable for its capacity to modulate a variety
of cellular behaviors (reviewed in Ref. 13). It is a member of a
superfamily of cytokines that includes bone morphogenetic proteins,
mullerian inhibitory substance, and activin, all of which regulate
cellular differentiation and matrix deposition in tissue repair. The
three isoforms of TGF-
(
1,
2, and
3) are structurally
related, with a high level of sequence homology and nearly identical
biological activities. Analysis of cDNAs has demonstrated that each is
initially synthesized as a larger precursor molecule containing the
mature form of TGF-
at the carboxy-terminal region, which is then
proteolytically cleaved, secreted as an inactive homodimer (latent
TGF-
), and then activated extracellularly before receptor
signaling.
1 may
play a central regulatory role in lung morphogenesis and
differentiation. TGF-
1 is expressed as early as day
11 of gestation in murine lung and colocalizes with
various extracellular matrix proteins including collagen types I and II
expressed at the epithelial-mesenchymal interfaces of stalks and clefts
of the developing lung (16). Immmunoreactive TGF-
1 increases between days 14 and
15 during differentiation of
primordial tubules into alveolar and bronchiolar ducts. Exogenous
TGF-
1 has been shown to inhibit branching morphogenesis in cultured
murine embryonic lung in a concentration-dependent manner (33).
Furthermore, both epithelial cell differentiation and lung sacculation
are delayed in transgenic mice bearing a chimeric gene composed of human SP-C gene promoter and porcine TGF-
1 cDNA (49), whereas targeted disruption of the TGF-
1 gene is associated with development of inflammatory infiltrates in the postnatal lung of homozygous knockout mice (36). These data suggest that although TGF-
1 may be
involved in normal growth and development of lung epithelial cells,
changes in spatial distribution and/or absolute levels of
expression can affect epithelial cell differentiation and organogenesis (37).
1 has been shown to have additional effects on
epithelial cell differentiation and surfactant component expression.
The arrested morphogenesis observed in the lungs from transgenic mice
bearing the SP-C-TGF-
1 gene was accompanied by decreased
immunostaining of the epithelial cells for surfactant proprotein
(proSP) C and Clara cell secretory proteins (49). Exogenous TGF-
1
added to alveolar type II cells isolated from the adult rat further
accelerated the culture-induced loss of SP-C mRNA (24). Inhibition of
SP-A expression has been observed in a human bronchiolar adenocarcinoma
cell line treated with exogenous TGF-
, but the mechanism for the
alteration of mRNA content is undefined.
1 has not been investigated in detail. Previously, inhibition of epidermal growth factor-induced increases in SP-A in
explants by a human platelet-derived preparation of TGF-
containing all three isoforms has been published (44).
1 on the expression of surfactant components and epithelial cell
ultrastructure in a cultured human fetal lung model. Our findings
indicate that TGF-
1 differentially downregulates the expression of
three SPs acting at the level of gene transcription, inhibits the
expression of a key regulatory enzyme for phospholipid synthesis, and
alters epithelial cell morphology. A preliminary report of this work
has been published (34).
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
1 was obtained from Genentech (San Francisco, CA)
and Collaborative Biomedical Products (Bedford, MA), and dexamethasone
was from Sigma (St. Louis, MO).
[32P]dCTP,
[32P]UTP, and
[methyl-3H]choline
(60 Ci/mmol) were purchased from NEN (Boston, MA).
Trans 35S label was obtained from ICN
Flow (Costa Mesa, CA). Polyclonal SP-A, SP-B, and proSP-C antisera
produced in rabbits have been previously described (4, 35, 39). Protein
A-agarose was obtained from Life Technologies (Bethesda, MD). All
culture medium was produced by the Cell Center Facility at the
University of Pennsylvania (Philadelphia).
All other reagents were electrophoretic grade and were purchased from Bio-Rad Laboratories (Hercules, CA) and/or Sigma.
Fetal Lung Explant Culture
Human fetal lung was obtained from second-trimester therapeutic abortions under protocols approved by the Committee in Human Research at Children's Hospital of Philadelphia and the Committee on Studies Involving Human Beings at the University of Pennsylvania. Distal lung parenchyma from fetuses 19-23 wk of gestation, previously screened for the presence of maternal infection, was chopped into 1-mm pieces and cultured as explants in serum-free Waymouth medium with and without dexamethasone (10 nM). TGF-Metabolic Labeling
Explants were starved in Met-Cys-free DMEM for 90 min, and metabolic labeling was done by the addition of trans 35S label-DMEM reaction mixture (100-200 µmCi/ml as methionine) for up to 4 h as previously described (35). After continuous labeling, samples were harvested by removal from the labeling medium and extensive rinsing in PBS containing protease inhibitors (5 µg/ml each of leupeptin, aprotinin, and pepstatin A). Harvested samples were frozen atmRNA Isolation
Total mRNA was isolated from harvested lung tissue with the TRIzol Reagent (GIBCO BRL, Gaithersburg, MD) according to the manufacturer's instructions. Briefly, tissue samples were homogenized in 1 ml of TRIzol Reagent and incubated for 5 min at 30°C. Phase separation was achieved by centrifuging samples at 12,000 g at 4°C for 15 min after the addition of 0.2 ml of chloroform. RNA was precipitated by further incubation and centrifugation after the addition of 0.5 ml of isopropanol. RNA pellets were subsequently washed with 75% ethanol and stored atAnalytic Methods
Western blotting. For SP-A, SP-B, and FAS analysis, lung explants harvested after 5 days in culture were sonicated and homogenized, then subjected to SDS-PAGE, and the separated proteins were transferred electrophoretically overnight as previously described (4, 35). After transfer, immunoblotting was performed separately with either primary anti-SP-A (1:5,000) or anti-SP-B (1:5,000) antisera and secondary goat anti-rabbit IgG antisera conjugated with horseradish peroxidase (1:10,000). Similarly, FAS protein was detected with a polyclonal anti-rat FAS antiserum (1:3,000) after explant proteins were separated on 5% SDS-PAGE. In each case, immunoreactivity was detected by enhanced chemiluminescence (ECL) with the ECL Detection Kit (Dupont-NEN, Bedford, MA) according to the manufacturer's instructions.Determination of protein content. Total protein content of lung tissue samples was determined by the Bradford (5) method with bovine IgG as the standard.
Quantitative immunoblot assay. Tissue levels of SP-A and SP-B were measured with a quantitative immunoblot assay as previously published (4). Briefly, fetal lung tissue was sonicated in a solution of protease inhibitors containing 10 mM benzamidine, 50 mM N-ethylmaleimide, and 10 mM phenylmethylsulfonyl fluoride. Samples of sonicated tissue (200-µl total volume) of known protein content were serially diluted in PBS, pH 7.5, and spotted onto nitrocellulose paper with a vacuum-assisted 96-well dot-blot apparatus (Bio-Rad). After the blots were dried and blocked with 2% milk, they were subsequently incubated with either primary anti-SP-A or anti-SP-B followed by a secondary goat anti-rabbit IgG-conjugated with horseradish peroxidase. Blots were washed extensively and exposed by ECL. The net density in each spot was determined by densitometric scanning with film exposures within the linear range. Results were normalized to SP-A or SP-B in a single pooled human surfactant prepared in our laboratory that was used as an internal standard. Assays were performed in duplicate on tissue from duplicate culture dishes, and protein content was read from the linear portion of the standard and unknown sample curves.
Immunoprecipitation. For proSP-C
analysis, radiolabeled fetal lung homogenates were solubilized in
buffer containing nonionic detergent and protease inhibitors and
immunoprecipitated with human proSP-C antiserum as previously described
(35). Proteins liberated by heating at 100°C for 15 min were
separated by one-dimensional 16.5% SDS-PAGE with a Tris-tricine buffer
system. Separated proteins were electrophoretically transferred to
0.2-mm nitrocellulose at 60 mA/cm2
for 12-18 h and visualized by autoradiography.
35S counts in specific
immunoprecipitated bands were quantitated by direct -scanning of the
blots with an AMBIS 4000 radioanalyzer (Scanolytics, San Diego, CA).
FAS activity. The activity of FAS (EC 2.3.1.85) was assayed by the incorporation of radioactive malonyl-CoA with the method of Roncari (30) as modified and described (12). Explants were sonicated in cold water and centrifuged at 13,000 rpm for 15 min, and aliquots of the supernatants were assayed by a 15-min incubation in the presence of 50 µM malonyl-CoA and 30 µM acetyl-CoA with 0.005 µCi [14C]malonyl-CoA in a 0.5-ml reaction volume. Enzyme activity, initiated by the addition of 50 µM NADPH, is expressed as nanomoles of malonyl-CoA utilized per minute per milligram of protein. All samples were assayed in duplicate.
Choline incorporation into phosphatidylcholine. To quantitate the rate of choline incorporation into phosphatidylcholine (PC), explants were cultured for the last 4 h in the presence of [3H]choline (1 µCi/ml medium), and then total lipid was extracted and PC was isolated by thin-layer chromatography on Whatman LK5D plates with chloroform-methanol-NH4OH (7 N; 60:35:5 vol/vol/vol) as previously described (11).
Quantitation of mRNA levels. Specific
mRNA content was determined by cDNA hybridization with Northern and
dot-blot analyses as previously described (28). Full-length human cDNA
inserts containing SP-A, SP-B, SP-C, and -actin have been previously used as probes by our group (23, 28). A human cDNA probe (clone Pg8)
containing a 2-kb fragment complementary to the 3'-end of the FAS
coding region (7) was a generous gift from Dr. D. Chalbos (Faculty of
Medicine, INSERM, Montpellier, France).
[32P]cDNAs for SP-A,
SP-B, SP-C, FAS, and
-actin were prepared from purified plasmid
inserts by labeling with
[32P]dCTP with a
random-primer labeling technique (Ready-To-Go Kit, Pharmacia,
Piscataway, NJ). Nitrocellulose blots were hybridized under high
stringency as previously published (23, 28) and then exposed to Kodak
XAR for 1-7 days at
70°C. Counts in specific mRNA bands
and dots were quantitated either by utilizing direct
-counting with
an AMBIS 4000 radioanalyzer (Scanolytics) or by densitometric scanning
of exposed film and quantitation with Quantity 1 (PDI, Huntington
Station, NY).
Nuclear transcription elongation (run-on) assay. The methods for the assay have been previously described (28). Nuclei were isolated from explants by homogenization and centrifugation. Nuclei (2.5 × 107) were resuspended in reaction buffer and incubated at 37°C for 30 min with 0.25 mM GTP, 0.25 mM CTP, 0.5 mM ATP, 100 µCi of [32P]UTP (3,000 Ci/mmol), and 40 U of RNase inhibitor. Digestion of the nuclei was done by successively adding RNase-free DNase I and proteinase K. RNA was prepared, and free nucleotides were removed with gel-filtration (Sephadex G-50) spin columns.
After linearization and denaturation, unlabeled human cDNAs for SP-A,
SP-B, SP-C, and -actin in Bluescript plasmid were applied to a
nitrocellulose filter strip (5 µg/dot) with a dot-blot apparatus. The
filters were hybridized with 2.5 × 106 counts/min of RNA at 42°C
for 4 days, washed under high-stringency conditions, and incubated with
RNase. The filters were exposed to X-ray film for 2-7 days at
70°C, and autoradiograms were quantified by densitometric
scanning.
Electron microscopy. Fetal lung explants were fixed in 2.5% glutaraldehyde-0.1 M sodium cacodylate (pH 7.2) for 3 h at 4°C, then rinsed in 0.1 M sodium cacodylate buffer (pH 7.2), and postfixed in 1% osmium tetroxide (12). After dehydration through a series of ethanols and propylene oxide, the tissues were embedded in epoxy resin. Ultrathin sections were cut with a diamond knife, contrasted with uranyl acetate and lead citrate, and evaluated in a JEOL CX100II transmission electron microscope operated at 80 kV.
TCA precipitation. Radiolabeled fetal lung tissue harvested at specified time points was sonicated and centrifuged at 14,000 g for 10 min. Aliquots of the supernatant were precipitated in 10% TCA, and the amount of label incorporated into acid-precipitable proteins was analyzed as previously described (35).
Statistics
Statistical analyses for group mean data were carried out with SigmaStat (Jandel Scientific). Comparisons of multiple groups by ANOVA were made with either Fisher's protected least significant difference or Student-Newman-Keuls test of normality. The level of significance was P < 0.05. ![]() |
RESULTS |
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Content of SPs
The treatment of cultured human fetal lung with TGF-
|
TGF-1 also inhibited the expression of SP-B in cultured lung.
Through a combination of transcriptional and posttranslational mechanisms, culture of human fetal lung induces both de novo synthesis and processing of 42-kDa proSP-B
(proSP-B42) to mature 8-kDa SP-B (SP-B8), and these effects are
markedly enhanced by inclusion of dexamethasone in the culture medium
(4). Under both control and dexamethasone conditions, TGF-
1 (100 ng/ml) completely blocked the expression of
SP-B8 (Fig.
2A).
Quantitative immunodot blotting demonstrated dose dependence of the
TGF-
1 effect on total SP-B immunoreactivity, with significant
inhibition at 10 ng/ml (Fig. 2B).
|
SP-C is a type II cell-specific protein considered to be a specific
marker of type II epithelial cell differentiation (27). Analysis of
mature SP-C expression has been hampered by its poor antigenicity, but
Solarin et al. (35) previously reported the usefulness of
proSP-C21 as a surrogate marker of
SP-C expression. Human fetal lung explants cultured for 5 days in the
presence and absence of dexamethasone were metabolically labeled with
[35S]Met-Cys and
immunoprecipitated with a proSP-C antiserum to identify synthesis of
the 21-kDa SP-C primary translation product. Dexamethasone induced a
marked upregulation of proSP-C21
expression compared with control cultures (Fig.
3A). The
addition of TGF-1 with dexamethasone on day
1 inhibited dexamethasone-stimulated synthesis of
proSP-C21. Quantitation of
35S in the immunoprecipitated
bands demonstrated that dexamethasone alone increased proSP-C
expression almost sevenfold compared with the control bands (Fig.
3B). Inhibition by TGF-
1 was dose
dependent, with a half-maximal response at ~10-20
ng/ml and complete inhibition occurring between 30 and 100 ng/ml. The
magnitude of the TGF-
1 effect on proSP-C expression increased with
exposure time (Table 1).
|
|
The observed effects of TGF-1 on SP expression were not a
consequence of a global inhibition of total protein synthesis by cytokine treatment. After 4 h of continuous labeling, incorporation of
[35S]Met-Cys into
total protein (measured as 35S
dpm/µg total protein) for dexamethasone- and TGF-
1-treated explants was determined and compared with control explants. There was
no significant difference in total
35S specific activity for control,
dexamethasone, or dexamethasone + TGF-
1 tissues (control, 31,191 ± 5,910 dpm/µg, n = 5;
dexamethasone, 37,058 ± 8,140 dpm/µg,
n = 5; dexamethasone + 10 ng/ml of
TGF-
1, 32,954 ± 3,107 dpm/µg,
n = 3; dexamethasone + 100 ng/ml of
TGF-
1, 42,578 ± 2,974 dpm/µg,
n = 3;
P > 0.05 for all groups vs. control group by ANOVA). Similarly, the percentage of TCA-precipitable 35S counts was unaffected by all
of the treatment combinations (data not shown).
SP mRNA Content and Transcription Rate
Figure 4 is a representative Northern blot analysis demonstrating the effects of TGF-
|
Quantitation of the downregulation of SP mRNA content by TGF-1 was
performed by analysis of data from multiple independent experiments
(Table 2). TGF-
1 at a concentration of
10 ng/ml substantially inhibited SP-A in both control and
dexamethasone-treated cultures. At the concentrations used, TGF-
1
alone was more inhibitory than dexamethasone for SP-A mRNA
(P < 0.05), and these responses were
not additive. TGF-
1 (10 ng/ml) also produced significant decreases
in the levels of both SP-B and SP-C mRNAs in cultures containing 10 nM
dexamethasone (P < 0.05 vs.
dexamethasone alone). Although SP-B mRNA was also downregulated by
TGF-
1 in the absence of dexamethasone, a significant effect on the
already low basal expression of SP-C mRNA could not be detected in
5-day control cultures.
|
The mechanism for TGF-1 inhibition of SP and SP message was further
investigated with nuclear run-on assays of gene transcription. Figure
5A shows
representative results for SP-A, SP-B, and SP-C. To avoid the effects
of prolonged exposure to TGF-
1 on cellular differentiation, explants
were cultured for 4 days in the presence of dexamethasone, and then
TGF-
1 was added during the final 24 h of culture. Under these
conditions, explants exposed to TGF-
1 showed ~80% inhibition of
the transcription rate for both SP-A and SP-C and ~50% inhibition
for SP-B (Fig. 5B).
|
Choline Incorporation and FAS Expression
Addition of exogenous TGF-
|
FAS represents an important regulatory enzyme in the de novo synthetic
pathway of surfactant PC (31). Western blots of lung homogenates from
explants treated with dexamethasone and/or TGF-1 were probed
with a polyclonal anti-FAS antibody, and immunoreactive protein content
was quantitated by densitometry. In parallel with data for choline
incorporation, dexamethasone stimulated, whereas TGF-
1 suppressed,
the level of FAS protein in lung homogenates (Table
4). Similar changes in FAS enzyme activity
were also observed in both control and dexamethasone-treated explants
(Table 4).
|
The changes in FAS protein content and enzyme activity were further
reflected in similar reductions in FAS mRNA content. By Northern blot
analysis, downregulation of the 9.0-kb FAS mRNA band was observed in
both control and dexamethasone-treated explants after the
administration of 10 ng/ml of TGF-1, with a higher dose (100 ng/ml)
completely abolishing the FAS mRNA signal (Fig. 6). Quantitation of the modulation of FAS
mRNA expression by TGF-
1 treatment was performed by dot-blot
analysis of RNA samples from multiple independent experiments and
indicated an equivalent percent decrease in both control and
dexamethasone-treated explants exposed to 10 ng/ml of
TGF-
1, and a greater response
to 100 ng/ml of TGF-
1 in the
presence of dexamethasone (Table 4).
|
Exogenous TGF-1 Alters Explant Ultrastructural
Morphology
|
The commensurate administration of TGF-1 at doses shown to block SP
and SP mRNA expression (10-100 ng/ml) markedly inhibited the
stimulatory effect of culture (Fig.
7D) and dexamethasone (Fig. 7,
E and
F) on lung maturation. Samples
treated with TGF-
1 showed a striking disruption of normal
differentiated type II epithelial cell characteristics, including a
loss of lamellar bodies and retention of cellular glycogen.
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DISCUSSION |
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TGF-1 has previously been shown to modulate normal lung development
and is implicated as a mediator in the pathogenesis of CLD of the
newborn (16, 20, 33, 49). In a series of experiments designed to test
the effect of TGF-
1 on human lung epithelial cell function and
differentiation, we administered recombinant human TGF-
1 to fetal
lung explants and evaluated the effects on cell-specific biochemical
and morphological markers of the type II cell phenotype. The results
presented here clearly demonstrate that TGF-
1 has profound effects
on the expression of both protein and lipid components of the
surfactant system. In addition, ultrastructural studies confirm a
marked effect of TGF-
1 on epithelial cell morphology.
In our experimental model, exogenous TGF-1 was delivered directly to
cultured lung explants, a stable, well-characterized model for testing
the effects of hormones, cytokines, and other agents on SP and lipid
metabolism (3, 4, 11, 12, 23, 34, 35, 40, 42-44). In the absence
of hormone and serum, the process of culturing lung explants for 5 days
leads to thinning of the mesenchyme, expansion of potential air spaces,
and differentiation of epithelial cells as characterized by the
development of microvilli and primordial lamellar bodies and by an
increased content of surfactant lipid and proteins (11). Our group and
others have found that the content of SP-A and SP-B increases (4, 23, 40, 43), whereas SP-C mRNA and proSP-C remain relatively low throughout
the 5-day culture period, even when late second-trimester explants are
utilized (23, 35, 43). For both SP-B and SP-C, dexamethasone treatment
increases mRNA content, protein synthesis, and posttranslational
processing of the resulting proproteins (4, 23, 35, 40, 43). This
enhanced expression of SP-B and SP-C is confined exclusively to the air
space epithelia that eventually undergo morphological differentiation
to type II cells. In contrast, SP-A, normally upregulated by culture
alone, is suppressed by continued exposure to dexamethasone, with a
biphasic dose-response curve (3, 42). Each of these patterns of SP
expression was reiterated by the current results reported here for
explants cultured in the absence of TGF-
1. Although a potential
limitation of this model is an inability to target specific cellular
subpopulations within the explant, similar responses to glucocorticoids
have been observed in vivo and support the physiological relevance of
the human fetal lung culture system as a model to test the effects of
cytokines.
The effect of TGF-1 on SP-A expression has been examined previously
with cultured H441 cells (41). At a dose of 1 ng/ml, TGF-
1
completely inhibited the expression of SP-A in this Clara cell-like
cell line. Our results showing inhibition of SP-A expression by
TGF-
1 in the explant system confirm this finding and provide additional evidence for the physiological relevance of this cytokine in
the modulation of SP expression. The higher doses required for maximal
inhibition of SP-A expression in the present studies are likely due to
an increased diffusion distance in the explants and/or the
presence of receptors on non-type II cells competing for TGF-
1.
Additionally, as yet undefined differences in signal transduction
events or TGF-receptor physiology between a transformed cell line and
human fetal lung may play a role.
In the present work, we demonstrated that TGF-1 could also inhibit
expression of the two hydrophobic SPs. In both the presence and absence
of glucocorticoid, TGF-
1 inhibited the expression of the mature
SP-B8. Because of the low basal
levels of proSP-C routinely observed in explant cultures lacking
exogenous steroids (35), we chose to study the effects of TGF-
1 on
SP-C under culture conditions containing dexamethasone. TGF-
1 (at
concentrations of 10-100 ng/ml) was inhibitory even in the
presence of stimulating doses of dexamethasone. Our results in the
cultured human fetal lung confirm previous findings for SP-C in vivo.
Zhou et al. (49) observed that lung-specific overexpression of TGF-
1
inhibited immunohistochemical staining for proSP-C in lungs of mice
containing a TGF-
1 transgene. In an in vitro system (primary adult
rat type II cell cultures), Maniscalco et al. (24) used in situ
hybridization to demonstrate an inhibitory effect of exogenous TGF-
1
on SP-C mRNA levels. Because of an inherent phenotypic instability of isolated type II cells, in which the levels of SP-C mRNA typically decline with time in culture on plastic, interpretation of the effects
of TGF-
1 on mRNA in that system should be made with caution.
The quantitative effects of TGF-1 on SP mRNA content paralleled the
decreases observed in the levels of protein and were accompanied by
similar inhibitions of the rates of gene transcription for each SP
(Fig. 5). The underlying molecular events of TGF-
1-induced inhibition of SP gene transcription are not fully characterized. Because the cultured human fetal lung is a complex multicellular model,
alterations of transcription rates after TGF-
1 administration could
involve indirect effects potentially mediated either by changes in
cellular composition or through changes induced in other cellular
constituents (e.g., fibroblasts). However, based on several lines of
evidence, it is more likely that the effects of TGF-
1 are a result
of its direct interaction with the epithelial cell. First, Torday and
Kourembanas (38) previously demonstrated that a TGF-
homologue
secreted by fetal lung fibroblasts can inhibit phospholipid synthesis
in cultured fetal type II cells. Second, TGF-
1 has been shown to
decrease SP-A and SP-B gene expression in H441 cells (41). Third, in
preliminary studies using plasmid transfections, we have found that
TGF-
1 (10 ng/ml), acting through a protein kinase C (calphostin
C-inhibitable) pathway, downregulates SP-B promoter activity in H441
cells (21). The effects of TGF-
1 on SP gene and SP expression are
reminiscent of those observed with
12-O-tetradecanoylphorbol 13-acetate
in both explants and H441 cells (28, 29). We have found that both
12-O-tetradecanoylphorbol 13-acetate
and TGF-
1 cause cytoplasmic trapping and loss of both thyroid
transcription factor-1 and hepatocyte nuclear factor-3 from the nucleus
of treated H441 cells (21, 22). It has previously been shown that these
transcription factors are important in SP gene expression and
epithelial cell differentiation (9, 46). Studies designed to address
the specific postreceptor pathway mediating these changes are currently
under further investigation.
We report the new observation that TGF-1 downregulates the
expression of FAS, an important regulatory enzyme in the synthetic pathway for the production of surfactant phospholipids (31, 37).
Similarly, choline incorporation into surfactant PC was markedly
decreased by TGF-
1. This finding is consistent with a previously
published study (38) using conditioned medium containing TGF homologues
from rat fetal lung fibroblasts to inhibit lipid synthesis by cultured
fetal rat type II cells. The decrease in lipid synthesis mediated by
TGF-
1 in this model likely reflects, at least in part, a functional
consequence of the decrease in FAS enzyme activity. The mechanism by
which TGF-
1 affects FAS is presently unknown but could reflect
either a direct action on FAS gene expression or an effect secondary to
TGF-
1-induced changes in other genes and/or epithelial cell
differentiation.
The effects of TGF-1 on lung epithelial differentiation and function
have not been widely studied. The overexpression of TGF-
1 in lung
epithelial cells of transgenic mice with the human SP-C promoter
induced a neonatal lethal phenotype, with delayed structural
development and inhibition of expression of two epithelial cell
markers, Clara cell secretory protein and SP-C (49). In our culture
system, exogenous TGF-
1 inhibited the normal ultrastructural maturation of the lung epithelium induced by culture and by
dexamethasone. Electron microscopy (Fig. 7) demonstrated that lung
explants cultured with TGF-
1 failed to develop the normal number or
appearance of lamellar bodies, a well-characterized phenotypic marker
of type II cells. In contrast to a lung bud system, tissue used in our
study is from midgestation, when branching morphogenesis is complete
and only cell differentiation is occurring (11). We observed that the
majority of the cells lining the lumens of potential air spaces from
the TGF-
1-treated explants contained substantially lower numbers of
lamellar bodies in contrast to tissue cultured with or without
dexamethasone. It is unlikely that the phenotypic changes in the
ultrastructural morphology observed in the presence of exogenous
TGF-
1 in this system are due to either a distortion of developmental
sequences during in vitro culture of the explants or a bias in the
acquisition of sections.
The lack of phenotypically normal type II cells could be secondary to
the deficiency of SP-B induced by TGF-1. In the syndrome of
congenital SP-B deficiency (10) as well as in the SP-B knockout mouse
(8), the phenotype is characterized by a lack of functional SP-B8 and associated abnormalities
in lipid metabolism, absence of lamellar bodies, disordered secretion,
and a block in posttranslational processing of SP-C. With the exception
of an increase in proSP-C processing intermediates, which would not be
expected given the downregulation of SP-C gene expression, many of
these same findings have been recapitulated in the TGF-
1-treated
lungs.
Alternatively, because TGF-1 is a multifunctional cytokine,
disruption of epithelial cell maturation and/or differentiation could occur apart from the inhibition of surfactant component expression. Since its discovery, TGF-
has been shown to function as
an autocrine/paracrine growth factor that exerts a variety of effects
on cellular functions, including regulation of cell proliferation,
differentiation, and extracellular matrix production (13). The cellular
response to TGF-
is dependent on cell type and culture conditions.
For cells of epithelial origin, TGF-
has been shown to generally act
as an inhibitor of growth. The proliferative response of alveolar type
II cells has been shown to be inhibited by TGF-
1 during
bleomycin-induced lung injury in rats (18). The effect of TGF-
on
differentiation is also variable; in some cases, the cytokine acts as a
suppresser of differentiation, whereas it stimulates the expression of
a differentiated phenotype in others. In the lung, detailed studies of
these effects are limited, but the administration of TGF-
has been
shown in vitro to induce squamous metaplasia (differentiation) in human bronchial epithelial cells (26). Thus the morphological and biochemical
effects seen with TGF-
1 administration could result from inhibition
of the normal progression of differentiation to type II cells during
culture or the promotion of differentiation toward another cell
phenotype (e.g., type I cell). The mechanism(s) by which TGF-
1
affects growth and differentiation is presently not defined. TGF-
type II receptors are present in the epithelial lining of the
developing airway (48). The biological effects occurring downstream
from the receptor-ligand complex may involve several possible signal
transduction mechanisms, including regulation of N-myc expression,
modulation of protein kinase activity, receptor-mediated tyrosine
phosphorylation, and Smad signaling (25).
Deficiency and/or dysfunction of the surfactant system has been
implicated in the pathogenesis of a variety of infectious, inflammatory, and toxin-mediated lung diseases (45). The findings that
TGF-1 inhibits surfactant components and type II cell
ultrastructural differentiation support the concept that this
multifunctional cytokine contributes to the pathophysiology of acquired
lung disease in part through its inhibitory effects on the lung
epithelium and the surfactant system. The doses of TGF-
1 used in
this study (10-100 ng/ml) are comparable to levels of the cytokine
that have been recovered early in the course (day
7) from the lungs of RDS patients who ultimately
developed CLD (20). Our data support further examination of the
possible role of surfactant deficiency in the pathogenesis of acquired
lung disease.
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ACKNOWLEDGEMENTS |
---|
We thank Scott Russo, John Gonzales, Sree Kumar, and Yue Ning for technical assistance. We thank Sylvia Decker for performing electron microscopy.
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FOOTNOTES |
---|
This work was supported by National Heart, Lung, and Blood Institute (NHLBI) Grant HL-02869 (to M. F. Beers); NHLBI Specialized Center of Research Grant 1-P50-HL-56401 (to P. Ballard, M. F. Beers, S. H. Guttentag, L. W. Gonzales, and J. Rosenbloom); National Institute of Child Health and Human Development Grant 5-P30-HD-28815 (to S. H. Guttentag); and Research Grants from the Pennsylvania Thoracic Society (to S. H. Guttentag) and the Philadelphia-Montgomery County American Lung Association (to M. F. Beers).
M. F. Beers is the recipient of a Clinician-Scientist Award from the American Heart Association.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: M. F. Beers, Univ. of Pennsylvania School of Medicine, 524 Johnson Pavilion, 36th and Hamilton Walk, Philadelphia, PA 19104-6068.
Received 21 January 1998; accepted in final form 30 July 1998.
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