1 Developmental Biology Program
and 2 Section of Molecular
Carcinogenesis, Newborn transforming growth factor
(TGF)-
transforming growth factor- LUNGS DEVELOP FROM AN OUTPOCKETING of the ventral
foregut endoderm surrounded by splanchnic mesenchyme (6). This process, from airway formation to saccular alveolarization, is regulated by
growth factors and cell-cell and cell-extracellular matrix (ECM)
interactions (36). Transforming growth factor (TGF)- Studies on TGF- In contrast to TGF- Homozygous TGF-
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
3-null mutant mice exhibit defects of palatogenesis and
pulmonary development. Glucocorticoids, which play a central role in
fetal lung maturation, have been postulated to mediate their
stimulatory effects on tropoelastin mRNA expression through TGF-
3 in
cultured lung fibroblasts. In the present study, we analyzed the
abnormally developed lungs in TGF-
3-null mutant mice and compared
the effects of glucocorticoids on gene expression and lung morphology
between TGF-
3 knockout and wild-type mice. Lungs of TGF-
3-null
mutant mice on embryonic day 18.5 did
not form normal saccular structures and had a thick mesenchyme between
terminal air spaces. Moreover, the number of surfactant protein
C-positive cells was decreased in TGF-
3-null mutant lungs.
Interestingly, glucocorticoids were able to promote lung maturation and
increased expression of both tropoelastin and fibronectin but decreased
the relative number of surfactant protein C-positive cells in fetal
lungs of both genotypes. This finding provides direct evidence that
glucocorticoid signaling in the lung can use alternative pathways and
can exert its effect without the presence of TGF-
3.
3; tropoelastin
expression
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
isoforms are
one key group of growth factors involved in lung organogenesis. The
TGF-
1, TGF-
2, and TGF-
3 isoforms all mediate a wide range of
biological effects including cell proliferation, differentiation, ECM
formation, and cell migration (19). However, the different
spatiotemporal expression patterns exhibited by the TGF-
isoforms
during embryogenesis and the distinct phenotypes of null mutants
demonstrate distinct roles in vivo (9, 11, 20, 22, 26, 31).
isoforms in lung morphogenesis
indicated that they have nonredundant roles during developmental
processes (12, 15, 37, 42). TGF-
1 is considered to be an inhibitory factor for lung development. In vitro experiments have shown that it
inhibits surfactant protein (SP) A and SP-C expression in fetal lungs
(15, 37). Exogenous TGF-
1 also inhibits branching morphogenesis in
embryonic lung culture (28). Overexpression of constitutively active
TGF-
1 in the distal lung epithelium arrests embryonic lung
sacculation and epithelial cell differentiation, delaying embryonic
lung development (42). However, an abnormal lung phenotype was not
observed in TGF-
1-null mutant fetuses, which has been attributed to
a maternal rescue effect (12).
1, much less is known about the role of TGF-
3
in lung development. TGF-
3-null mutants show abnormal lung
development and defective palatogenesis, leading to death within 24 h
after birth (9, 20). The observed abnormal lung morphology in
TGF-
3-deficient [TGF-
3(
/
)]
newborn mice includes alveolar hypoplasia, lack of septal
formation, mesenchymal thickening, and a decreased number of type II
alveolar epithelial cells (9). These findings are reminiscent of
immature lungs, which can cause newborn respiratory distress syndrome
in human premature infants. A study (35) on the expression of TGF-
3
from primary cultures of fetal lung fibroblasts isolated from different
embryonic stages has shown a peak at an early canalicular stage.
TGF-
3 is also the most abundant glucocorticoid-induced transcript in
fetal rat lung fibroblasts (35). Blocking endogenous TGF-
3
production by antisense oligonucleotides or its activity by
neutralizing antibody abrogates the stimulatory effect of
glucocorticoids on tropoelastin mRNA expression by cultured fetal lung
fibroblasts (40). Because glucocorticoids are known to accelerate
pulmonary maturation and are used clinically to prevent respiratory
distress syndrome in premature human infants, it is important to
understand the molecular and cellular mechanisms of action of
glucocorticoids in lung development and their connection to TGF-
3 in
vivo. This is the focus of the present study.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
3 Knockout Mice
3(+/
) mice were previously generated in our
laboratory (9). Homozygous TGF-
3(
/
) fetal mice were
produced by timed mating between TGF-
3 heterozygous male and female
mice. The finding of a vaginal plug was counted as embryonic
day (ED) 0. The pregnant mice were killed on
ED18.5, and the fetuses were removed by cesarean section. The fetal
lung was excised en bloc and dissected into each individual lobe under
a dissecting microscope. Different lobes were processed for either
fixation or RNA extraction. Genomic DNA was isolated from a part of the
tail, and the genotype was confirmed by Southern blot analysis (9).
Preparation of Tissue Sections
The superior lobe of the right lung was fixed for 4 h in 4% paraformaldehyde-PBS at 4°C. The fixed tissues were then dehydrated in ethanol and embedded in paraffin. Coronal sections were cut at 6 µm thickness and mounted on Superfrost glass slides (Fisher Scientific, Pittsburgh, PA). The slides were dried at 37°C overnight and 60°C for 1 h. The sections across the middle part of the lobe were chosen for this study. Hematoxylin and eosin staining was used to study the histological structure of different sections. Comparison between wild-type and TGF-Morphometry
The area of terminal respiratory air spaces was measured with SigmaScan version 3.1 (Jandel Scientific, Corte Madera, CA). Multiple measurements were performed on randomly selected 0.04-mm2 fields located at the distal part of the fetal lungs. The proportion of lung comprising terminal air spaces is presented as a percentage of the total area of the lung section. The mean ± SD was obtained from sections of three individual fetal lungs of wild-type or TGF-Immunohistochemistry
Lung sections were first deparaffinized in xylene, followed by rehydration in series of different concentrations of ethanol. The endogenous hydrogen peroxidase was quenched with 3% H2O2 in methanol for 10 min. Zymed Histostain-Plus (Zymed, South San Francisco, CA) was used for the following staining procedures.SP-C immunohistochemical staining. Rabbit anti-proSP-C polyclonal antibody was a generous gift from Dr. Jeffrey Whitsett (Children's Hospital Research Foundation, Cincinnati, OH) (34). The sections were incubated with antibody diluted 1:1,000 in PBS-0.2% Triton X-100 overnight at 4°C. Positively stained epithelial cells were counted, and a comparison between wild-type control and null mutant mice was performed.
Anti-fibronectin immunostaining. A rabbit anti-fibronectin polyclonal antibody was purchased from Sigma (St. Louis, MO). The sections were incubated with antibody diluted 1:1,000 for 1 h at room temperature.
Elastin staining. Hart's elastin staining method was used (14). Briefly, the deparaffinized section was first treated in potassium permanganate for 30 min followed by a rinse in a 5% oxalic acid solution and then stained in resorcin-fuchsin solution overnight and finally in van Gieson's solution for 1 min. The number of positively stained fibers and their intensity were compared between wild-type control and null mutant mice.
Total RNA Isolation and RT
The lungs were first dissected into each individual lobe. To eliminate the contribution of large blood vessels and large bronchi, approximately one-third of the peripheral portion of the lung was dissected and immediately frozen in liquid nitrogen. The tissues were further processed for total RNA isolation with a Qiagen RNeasy kit (Qiagen, Santa Clarita, CA). The quality of isolated RNA was checked by agarose gel electrophoresis before the RT reaction.About 200 ng of total RNA in 25 µl containing 1.25 µg of oligo(dT) (Pharmacia), 50 mM Tris · HCl, pH 8.0, 70 mM KCl, 10 mM MgCl2, 1 mM deoxynucleotide triphosphates, 10 mM dithiothreitol, 40 U of RNasin ribonuclease inhibitor (Promega, Madison, WI), and 1,000 U of Moloney murine leukemia virus reverse transcriptase (Life Technologies, Gaithersburg, MD) were used for each RT reaction at 37°C for 90 min. The product of the RT was diluted fivefold and applied to competitive PCR.
Primers and Competitive RT-PCR
The competitive RT-PCR method has been previously described (41). For tropoelastin competitive PCR, a 291-bp fragment of mouse tropoelastin cDNA was amplified by PCR with the primers 5'-TGCCAAAGCTGCTGCTAAGGCT-3' and 5'-AGTCCAAAGCCAGGTCTTGCTG-3'. In addition, a 344-bp competitor template fragment was constructed from v-erbB DNA by PCR with a pair of composite primers, which were attached with the same nucleotide sequences of tropoelastin primers at the ends. In this way, the tropoelastin-specific primer sequences were incorporated into both ends of the competitive cDNA template, which can also be amplified with the same pair of tropoelastin primers.Primers and competitor templates for -actin, fibronectin, and SP-C
genes were designed in a similar way as previously described (39).
Competitive RT-PCRs were performed on cDNA samples described above with
known amounts of competitive templates. The reaction mixture contained
10 mM Tris · HCl (pH 9.4), 50 mM KCl, 2 mM
MgCl2, 0.01% gelatin, 0.1%
Triton X-100, 20 pmol of the primer sets, 100 µM deoxynucleotide
triphosphates, and 0.5 U of Advantage Klentech DNA polymerase
(Clontech, Palo Alto, CA) in a total volume of 50 µl. PCR
amplification was carried out in a Robocycler (Stratagene, La Jolla,
CA) with a modification of a previously described assay (41) for the
TGF- type II receptor (35 cycles of denaturation at 94°C for 2 min, annealing at 62°C for 2 min, and extension at 72°C for 2 min).
For quantitation, a standard curve was always made with a series of
cDNA standard dilutions as samples (Fig. 1,
A and
B). The concentration
of cDNA was determined spectrophotometrically. As an internal control,
-actin competitive PCR was performed on the same samples. As a
negative control for mouse genomic DNA or v-erbB DNA,
un-reverse-transcribed total RNA and adenoviral DNA were also included
in the competitive PCR assays.
|
Gel Electrophoresis and Quantitation of PCR Products
A 3% agarose gel (3:1 mixture of NuSieve and SeaKem, FMC BioProducts, Rockland, ME) was used to separate target and competitor PCR products. The intensity of each band was determined by densitometric analysis with Image- Quant band-analyzing software (Molecular Dynamics, Sunnyvale, CA). RNA samples from three individual wild-type or TGF-Maternal Dexamethasone Treatment of Mice
Pregnant mice were injected intramuscularly with dexamethasone or saline consecutively on ED15.5 and ED16.5. The dosage of water-soluble dexamethasone (Sigma) was 2 µg/g body wt. ![]() |
RESULTS |
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Morphological Characterization of TGF-3
Knockout Fetal Lungs on ED18.5
|
Because TGF-3 is a possible candidate for the mediating effects of
glucocorticoids in fetal lungs, lack of TGF-
3 function was predicted
to cause insensitivity to glucocorticoid treatment. To test this
hypothesis, we compared the morphology of ED18.5 fetal lungs with and
without maternal administration of dexamethasone on ED15.5 and ED16.5.
Dexamethasone treatment enhanced saccular formation in TGF-
3
knockout fetal lungs (Fig. 2, G and
H) as well as in wild-type fetal
lungs (Fig. 2, C and
D). The proportion of terminal air
space area was increased in both genotypes after dexamethasone
treatment (65 ± 8% in wild-type fetal lungs,
P < 0.025; 54 ± 6% in TGF-
3
knockout fetal lungs, P < 0.005).
However, the effect was greater in wild-type fetal lungs, which
exhibited morphology approaching the postnatal alveolar stage.
Decreased Expression of Tropoelastin and Fibronectin in
TGF-3-Null Fetal Lungs
Tropoelastin mRNA expression in TGF-3 knockout fetal lungs was
~70% of the expression level in wild-type control lungs
(P < 0.05; Fig. 1,
C and
D). However, maternal administration
of dexamethasone resulted not only in a 300% increase in tropoelastin expression compared with untreated control wild-type fetal lungs (P < 0.05) but also in a 230%
increase in tropoelastin expression compared with untreated TGF-
3
knockout fetal lungs (P < 0.05). This result was confirmed by Northern blot analysis (data not shown).
Therefore, TGF-
3 is not absolutely required for
dexamethasone-induced tropoelastin expression. Because tropoelastin is
the monomeric form of elastin that constitutes elastin fibers, elastin
fibers of ECM components were visualized by elastin-specific staining (Fig. 3). In wild-type fetal lungs on
ED18.5, elastin fibers could be easily seen around mature saccular
structures and, occasionally, on the tips of the septal buds. With
dexamethasone treatment, elastin fibers appeared thicker, and bundles
of elastin fibers were easily seen in areas of septal formation.
Compared with wild-type fetal lungs, the amount of elastin fibers in
TGF-
3 knockout fetal lungs was decreased. Also, the fibers were
thinner and appeared in fewer mature saccules. In contrast, thick
bundles of elastin fibers were seen in dexamethasone-treated TGF-
3
knockout fetal lungs.
|
Besides tropoelastin, expression of fibronectin was slightly reduced in
TGF-3 knockout fetal lungs compared with wild-type control lungs
(Fig. 4). Treatment with dexamethasone
resulted in a 60-80% increase in fibronectin expression in both
genotypes. Immunostaining of fetal lung sections for fibronectin did
not show any striking differences in the distribution pattern of
fibronectin between wild-type and TGF-
3 knockout samples (data not
shown).
|
Type II Epithelial Cells and SP-C Expression
Differentiation of epithelial cells is characteristic of fetal lungs at the saccular stage. Type II epithelial cells synthesize and secrete SPs such as SP-C. Using a quantitative competitive RT-PCR method, we found that SP-C expression in TGF-
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|
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DISCUSSION |
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Role of TGF-3 in Fetal Pulmonary Development
The abnormal morphology of TGF-3 knockout fetal lungs on ED18.5 was
not a simple arrest of pulmonary development at a particular stage.
Parts of nearly mature saccular structures can still be seen in
TGF-
3 knockout lungs, but the generally increased thickness of the
mesenchyme between the terminal air spaces with more layers of cells
indicates an abnormal phenotype. Normally during the late prenatal
period, alveolar air space area gradually increases and mesenchyme
thickness decreases. Treatment with dexamethasone in the late
pseudoglandular and early canalicular stages promoted saccular
structure formation in both wild-type and TGF-
3 knockout mice.
However, the morphological maturation of knockout fetal lungs still
appeared to be retarded in comparison with that of wild-type fetal
lungs after maternal dexamethasone treatment. This indicates that
dexamethasone cannot fully restore the phenotype caused by TGF-
3
deficiency, which is consistent with the observation that morphometric
changes in the lung parenchyma after glucocorticoid exposure are not
analogous to normal maturation changes (38).
The mechanism of mesenchymal thinning is poorly understood. In
wild-type late-gestation lungs, the number of apoptotic cells is very
low, and, therefore, it is very unlikely that mesenchymal thinning
results from programmed cell death (3, 19). The hypercellular lung
phenotype, with a thick mesenchyme and poorly developed prealveolar
spaces, has also been described in corticotropin-releasing hormone
(CRH)-deficient mice (18). The lethal phenotype of these mice can be
rescued by prenatal administration of glucocorticoids; this provides
further evidence for the importance of glucocorticoid signaling in
late-term pulmonary maturation (18, 19). In wild-type mice, mesenchymal
cell proliferation ceases during the late-gestational period, whereas
in CRH-deficient mice, cell proliferation continues (19). The striking
similarity between the pulmonary phenotypes of the CRH-deficient and
TGF-3-deficient mice suggests that the impaired lung maturation in
TGF-
3-null mutant mice is caused by a defect in glucocorticoid
signaling that leads to the continued proliferation of mesenchymal
cells. The fact that the TGF-
3-null mutant phenotype can, at least
partially, be restored by exogenous administration of glucocorticoids
would indicate that glucocorticoid-induced lung maturation in
TGF-
3-null mutant mice works above certain threshold; i.e., in these
mice, the endogenous glucocorticoid level is not sufficient to reduce
mesenchymal cell proliferation.
TGF-3 Regulates Expression of Tropoelastin and
Fibronectin in Fetal Lungs
In addition to tropoelastin, another important ECM component,
fibronectin, was found to be expressed at a somewhat lower level in
TGF-3 knockout fetal lungs. Fibronectin is an adhesive substrate in
the ECM that can bind to integrin receptors on cell surfaces to affect
cell proliferation, differentiation, migration, and apoptosis. The
expression of fibronectin in murine embryonic lungs has been detected
in the mesenchyme and parabronchial cells at the pseudoglandular stage
(23). Intense staining of fibronectin was observed at the branching
points of developing airways in wild-type fetal lungs. Blockade of
fibronectin binding to its integrin receptors in cultured murine lung
explants inhibited branching morphogenesis (24, 25). In the late stages
of pulmonary development, fibronectin expression is normally decreased;
however, it has been shown that fibronectin promotes fetal lung
vasculogenesis and angiogenesis in vitro (7, 8). Whether the reduced
fibronectin expression is related to the observed phenotypes in
TGF-
3-null mutant mice requires further study.
TGF-3 Regulates Epithelial Cell
Differentiation
In summary, our in vivo experimental data from the TGF-3 knockout
mouse model indicates that TGF-
3 is essential for the morphological
and functional maturation of the fetal lung and the normal expression
levels of tropoelastin, fibronectin, and SP-C during lung development.
In concordance with the studies of others (19), exogenous
glucocorticoids enhance the fetal lung maturation and expression of
tropoelastin in wild-type mice. Moreover, the opposite response of
tropoelastin and SP-C expression to glucocorticoid treatment suggests a
dissociation between the processes involved in connective tissue
remodeling and epithelial cell differentiation. Our present results
demonstrate similar, albeit less pronounced, changes in TGF-
3-null
mutant mice and would argue that glucocorticoid signaling in the lung
does not absolutely require TGF-
3 in vivo.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Richard A. Pierce for initial involvement and discussions during the beginning stage of this study and Dr. Jeffrey A. Whitsett (Children's Hospital Research Foundation, Cincinnati, OH) for providing the anti-surfactant protein C antibody. We also thank Pablo Bringas and Valentino Santos for guidance in elastin staining and Dr. Yang Chai for assistance in morphometric measurement.
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FOOTNOTES |
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This study was supported by a Childrens Hospital Los Angeles Research Institute Fellowship (to W. Shi) and National Heart, Lung, and Blood Institute Grants P01-HL-60231 (to J. Groffen, N. Heisterkamp, and D. Warburton), HL-44060, and HL-44977 (both to D. Warburton).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: V. Kaartinen, Developmental Biology Program, Dept. of Pathology, MS #54, Childrens Hospital Los Angeles, 4650 Sunset Blvd., Los Angeles, CA 90027 (E-mail: kaartine{at}hsc.usc.edu).
Received 11 March 1999; accepted in final form 27 July 1999.
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