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Division of Newborn Medicine, The Floating Hospital for Children, New England Medical Center, Tufts University, Boston, Massachusetts 02111
Address all correspondence and requests for reprints to: Christiane E. L. Dammann, Division of Newborn Medicine, The Floating Hospital for Children, New England Medical Center, Tufts University, Boston, Massachusetts 02111. E-mail: christiane.dammann{at}es.nemc.org
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Introduction |
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Growth factors and hormones are important regulators of fetal lung cell growth and maturation (4, 5). Epidermal growth factor (EGF) is a potent mitogen that stimulates DNA synthesis and cell proliferation in fetal lung fibroblasts (6). EGF also affects the progression of cell differentiation (7, 8). The effect of EGF is mediated through binding to its receptor, a 170-kDa transmembrane glycosylated phosphoprotein with intrinsic tyrosine kinase activity (9).
EGF stimulates fetal lung maturation both in vivo (10, 11, 12) and in vitro (5, 13, 14, 15, 16, 17, 18). However, EGF fails to directly stimulate surfactant synthesis in fetal (15, 16) and adult (19) type II cells. Thus, type II cell maturation is determined by mesenchymal-epithelial cell communication (3, 20). The process of cell-cell communication is not clearly understood, but there is evidence that the control of cell-cell communication may involve the combined effects of hormones and growth factors, which regulate the production by fibroblasts of a soluble factor that acts on type II cells (reviewed by Smith, Ref.21). One possible mediator of this process is the fibroblast-pneumonocyte factor, an apparent protein released by fibroblasts, which mediates hormonal signals between the fetal lung mesenchyme and epithelial cells (21). The regulation of the EGF-receptor (EGF-R) may play an important role in this process (15). Fetal lung fibroblasts have larger amounts of EGF-R than isolated type II cells (19). The EGF-R is developmentally regulated during late gestation. Fetal lung fibroblasts exhibit maximal EGF-R binding at the time of the onset of augmented surfactant synthesis (13, 22).
In this study, we hypothesized that EGF, cortisol, and RA (known as positive regulators of lung development), as well as DHT and TGF-ß1 (known as negative regulators), affect EGF-R binding and EGF-R protein abundance in late gestation fetal lung fibroblasts. We also hypothesized that these effects are sex specific and different at different stages of lung cell maturation. We tested this hypothesis in fetal rat lung fibroblasts at d17, d19, and d21 (term = d22), an interval in late gestation when fetal rat lung fibroblast EGF-R binding peaks and then declines (22).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Fetal rat lung fibroblast cultures
Fibroblast cell cultures were prepared using procedures we have
previously described (15, 23). Time-dated pregnant rats were killed on
d17, d19, or d21 of gestation (term d22) by CO2 inhalation.
Usually, four to five dams were used per preparation. The uterus was
removed under sterile conditions by laparotomy and kept on ice. Fetuses
were sexed (24) and kept in DMEM on ice. The lungs were removed en bloc
under a laminar air flow hood, put into sterile HBSS, pooled separately
according to sex, and minced into 1 mm3 pieces with a razor
blade. The minced lungs were dissociated in HBSS containing DNAse and
250 mg trypsin in a 37 C water bath using a stirring bar to disrupt the
tissue physically for 10–12 min. The reaction was stopped by adding
ice-cold DMEM containing 10% charcoal-stripped calf serum
(CS-) [treatment with activated charcoal removes steroid
hormones (25) and EGF (17)]. The cells were filtered through a sterile
70-µm cell strainer and centrifuged at 650 x g for
10 min at 4 C. The pellet was resuspended in DMEM containing 10%
CS- and plated in six-well plates for 60 min at 37 C to
allow for differential adherence of lung fibroblasts. After this, the
medium was changed to either DMEM with 10% charcoal-stripped FCS
(FCS-) (controls), or DMEM with 10% FCS-
containing one of the following treatments: EGF (tissue culture grade;
10 ng/ml), cortisol (10-7 M),
all-trans-RA (10-5 M), DHT
(10-7 M), or TGF-ß1 (2 ng/ml). Media were
changed every 24 h. When cultures reached 90–95% confluence
(generally after 5 days) the 125I-EGF binding assay or
Western blot analysis was performed. In additional experiments,
dose-response effects of each treatment on EGF-R binding were studied
in d19 female and male fetal rat lung fibroblasts.
125I-EGF binding assay
EGF-R binding was measured using methods previously described
(22, 26, 27) and adopted by us. For each treatment condition, confluent
fibroblast cultures were washed twice with 2 ml prewarmed DMEM to
remove the treatment and remaining FCS-, then incubated
further for 30 min at room temperature or in the case of EGF treated
cultures for 4 h at 37 C to let the EGF-R reconstitute. We have
previously shown that increasing doses of unlabeled EGF competitively
decrease 125I-EGF binding to the EGF-R (22). Therefore, we
measured binding of 125I-radiolabeled EGF to sites other
than the EGF-R (nonspecific binding) (28, 29) in three wells of the
six-well plate containing 1 ml DMEM by incubating with a 500-fold
excess (200 ng/ml) unlabeled EGF (receptor grade) for 20 min at room
temperature. At the end of this incubation period, 0.4 ng/ml
125I-EGF was added to all six wells. After 60 min
incubation at room temperature, the cells were washed three times with
2 ml ice-cold PBS, pH 7.4, to remove the unbound radiolabeled EGF and
scraped in 1 ml PBS. Radioactivity was measured using a gamma counter.
In each experiment, specific EGF binding was determined in two or three
wells by subtracting the radioactivity of the mean nonspecific binding
(average binding in triplicate wells preincubated with unlabeled EGF)
from the total binding (binding in wells not preincubated with
unlabeled EGF). DNA was measured as deoxyribose content in duplicate
aliquots (30). The mean of the nonspecific binding was <8% of total
binding.
Results of the binding studies are given either as pg bound EGF per nmol DNA or as percent of the binding in sex- and experiment-specific controls. Hereafter, these are referred to as "absolute" and "relative" binding, respectively.
Western blotting of the EGF-R
Western blot analyses were performed in additional experiments
using fetal rat lung fibroblasts of each sex at d17 and d19. Confluent
fibroblast cultures were harvested by cell scraping in PBS, pH 7.4,
containing leupeptin 2 µg/ml, aprotinin 1 µg/ml, antipain 2
µg/ml, and orthovanadate 37 µg/ml. After sonication, aliquots were
removed and used to measure total protein concentration (31). The
remainder of each sample was then diluted with an adequate volume of
Laemmli buffer (32), boiled for 5 min at 100 C and stored at -70 C for
later analysis. The samples (20 µg total protein/35 µl) and
molecular weight markers were electrophoresed on 7.5% poly-acrylamide
SDS gel. After protein transfer by electroblotting to a PVDF membrane,
nonspecific binding was blocked in dH2O with 1% gelatin
for 2 h at room temperature. Blots were then incubated with 1–2.5
µg/ml of a polyclonal sheep antihuman EGF-R antibody overnight at 4
C. The blots were washed three times in TBST buffer [10 mM
Tris base, 150 mM NaCl, 0.05% Tween 20 (pH 7.4)] and
exposed to the secondary antibody (alkaline phosphatase conjugated
donkey antisheep IgG) for 1 h at room temperature. Blots were
washed three times with TBST and visualized with alkaline phosphatase
buffer containing BCIP and NBT. Western blot membranes were scanned and
quantified thereafter by computerized densitometry.
Data analysis
Statistical analyses were performed using the two-sample
t test or ANOVA. A P value <0.05 was considered
statistically significant. Bonferroni correction for multiple
comparison was made where appropriate. In such instances, statistical
significance is indicated by a P value <0.01.
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Results |
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). EGF-binding was
significantly higher in fetal rat lung fibroblasts on d19 when compared
with d17. Overall, binding was lower in male than female cells, but
this difference was not statistically significant.
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).
Although the physiologic exposure of the fetal lung to EGF via amniotic
fluid may be somewhat lower (33), we chose a dose of 10 ng/ml for the
subsequent experiments, because of the proven efficacy of this dose on
lung maturation in vitro (13, 15, 16).
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). The
dose we used for the subsequent experiments (10-7
M) is comparable with the cortisol concentration in human
amniotic fluid at 28 weeks of gestation (2), whereas the serum level of
cortisol is 10-8 M in the human fetus in
midgestation and rises until term (34).
As opposed to EGF and cortisol, increasing doses of RA significantly
increased the relative EGF-R binding in females. In male cells, binding
peaked at a concentration of 10-8 M. This
increase was statistically significant. Sex differences were
significant in the 10-10–10-8 M
range only (Table 1). The dose (10-5 M) we
used for the subsequent experiments is similar to that of retinol in
midgestation amniotic fluid (35). A dose of
10-6–10-5 M has been used in
other studies to stimulate fetal lung surfactant synthesis (5, 36, 37).
The negative regulators DHT and TGF-ß1 significantly altered EGF-R binding in a sex-dependent and dose-dependent manner. DHT treatment of female cells caused a significant increase in specific EGF-R binding with increasing concentrations. Although DHT stimulated EGF-R binding in male cells, the response fell slightly short of statistical significance. A female-male difference was significant at a dose of 10-9 M and 10-6 M. Although the physiologic levels of dihydrotestosterone in amniotic fluid or plasma (38, 39) may be somewhat lower, we chose a dose of 10-7 M for the subsequent experiments. Similar doses have been previously used in studies of lung surfactant synthesis (40, 41, 42).
Treatment with increasing doses of TGF-ß1 caused little change in
EGF-R binding in female cells (n.s.), and a significant decrease in
EGF-R binding in male cells. The female-male difference was significant
at lower doses only (Table 1). We used a dose of 2 ng/ml for the
subsequent experiments, because a similar dose has been shown to
inhibit surfactant synthesis (14) and the physiological concentration
of bioactive TGF-ß in the amniotic fluid is in the same range
(43).
Influence of gestational age on the response of EGF-R binding and
EGF-R protein abundance to treatment
The alterations in EGF-R binding by treatment with EGF (10 ng/ml),
cortisol (10-7 M), RA (10-5
M), DHT (10-7 M), or TGF-ß1 (2
ng/ml) compared with untreated controls are shown in Fig. 2A (for day 17 fibroblasts), Fig. 3A (for day 19 fibroblasts), and Fig. 4 (for day 21 fibroblasts). Virtually all
treatments appeared to increase the relative EGF-R binding in d17 fetal
lung fibroblast cultures when compared with experiment- and
sex-specific controls (Fig. 2A). The relative increase was minimal and
not significant for EGF (both sexes) and TGF-ß1 (male cells only).
Larger apparent increases (cortisol, both sexes, TGF-ß1 in female
cells) also were not significant. DHT increased EGF-R binding, although
this result did not achieve nominal statistical significance. RA
increased EGF-R binding significantly in both females (430% of
controls) and males (510% of controls). There were no statistically
significant female-male differences.
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) and male
fibroblasts (data not shown) after treatment with EGF, cortisol, RA,
DHT, or TGF-ß1 were similar to the corresponding EGF-R binding
patterns on d17 (Fig. 2A). Computerized densitometry of the d17 Western
blot membranes (Fig. 2C) confirmed these results. These data indicate
that changes in EGF-R binding after treatment were associated with
similar changes in receptor protein abundance.
In d19 fetal lung fibroblasts (Fig. 3A) treatment with RA (both sexes)
and DHT (female cells only) resulted in statistically significant
increases in relative EGF-R binding. Cortisol treatment caused a
statistically significant decrease in females but no effect in male
cells. The female-male difference after cortisol treatment was
statistically significant. Treatment with EGF or TGF-ß1 resulted in a
minimal effect on EGF-R binding.
Western blot analysis (Fig. 3B) and computerized densitometry (Fig. 3C)
were again performed to test if the observed changes were related to
changes in EGF-R protein abundance. These results confirmed the results
of the binding assay at that particular gestational day, again
indicating that EGF-R protein abundance changed in accordance with
changes in binding.
At d21 (Fig. 4), RA stimulated EGF-R binding in both sexes, but this
was statistically significant in female cells only. Stimulation of
EGF-R binding by TGF-ß1 was statistically significant in female
cells, but again males fell just short of significance. DHT appeared to
stimulate binding, but this did not reach statistical significance. On
the other hand, EGF treatment appeared to cause a modest decrease and
cortisol a modest increase in EGF-R binding. Neither of these small
effects nor female-male differences achieved statistical
significance.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In an effort to achieve physiologic relevance of the doses of the hormones and growth factors used in this study, we adjusted the treatment concentration to their concentration in the fetal circulation. Where no data were available for circulatory concentrations, concentration in the amniotic fluid during that particular time in gestation were used instead. Even though the net flow of amniotic fluid might be out of the lung, there is bulk flow of amniotic fluid in and out of the lung which increases with advancing gestation (49, 50). Thus, we consider amniotic fluid concentrations to be part of the physiological environment of the fetal lung cells in utero.
The effect of EGF treatment on EGF-R binding and EGF-R protein abundance in late fetal lung fibroblasts was surprisingly low. This is in contrast to the finding of an increase in mesenchymal EGF production during development (48). In addition, EGF advances other aspects of the fibroblast (15) and type II cell maturation (17) later in gestation.
The clinical role of antenatal corticoid treatment in the prevention of severe respiratory distress syndrome in the preterm infants is well known (51). Maternal administration of glucocorticoids increases fetal surfactant production. Direct exposure of fetal type II cells to glucocorticoids has minimal effect on surfactant synthesis, but when d19 fetal rat lung fibroblasts are exposed to dexamethasone, and conditioned medium is prepared, the conditioned medium stimulates DSPC synthesis in type II cells (3, 16). Thus, a mechanism involving the mesenchymal-epithelial interactions is very important for the type II cell maturation (3). It has been uncertain whether cortisol influences this cell-cell communication process through changes in EGF-R. Our data indicates that at gestational days with a high endogenous EGF-R binding, cortisol seems to have a minimal additional effect on binding and protein abundance.
In this study, retinoic acid was the treatment that consistently caused large increases in EGF-R binding and protein quantity. The marked increase in EGF-R binding after RA treatment suggests that fetal lung fibroblasts are most sensitive to RA treatment in late gestation when the concentration of retinoids in the fetal rat lung has already reached the maximum (52). The effect of RA on epithelial maturation is mediated by cell-cell communication (48), and the EGF-R in fibroblasts may play a role in this signaling pathway.
The human fetal lung expresses androgen receptors and activates testosterone by converting it to its more active and specific form DHT (53). DHT treatment is usually viewed as an inhibitor of lung cell maturation (41). Continuous in vivo or in vitro androgen exposure leads to delayed cell maturation only when initiated early in lung development (25, 54, 55). In contrast, this study shows that DHT treatment of fibroblasts cultured from late gestation lungs resulted in an increase in EGF-R binding. Female fibroblasts were more responsive to DHT than males. The reasons underlying the observed female-male differences to DHT treatment on EGF-R binding are unclear. There is a known female-male difference in physiologic circulating androgen concentration during fetal development (39, 56) which becomes maximal at the gestation studied here, but whether this is also associated with a female-male difference in androgen receptors is unknown. However, the sex difference we observed may reflect the sex specific differential lung maturation pattern over gestation (2, 15, 23, 41, 54).
The role of TGF-ß1 in lung development is still incompletely understood. TGF-ß1 inhibits the development of lung cell maturation at specific gestational stages (14, 20, 47). In contrast, in this study TGF-ß1 treatment increased EGF-R binding in lung fibroblasts cultured from late gestation.
Most studies of the modulation of EGF-R have been done using cell lines. In this study we used primary fetal lung cells to obtain information about the possible modulation of maturation at specific windows of development during gestation and thereby learn more about the mechanisms by which the EGF-R may act to regulate cell maturation in vitro. Another advantage of our approach was the use of fibroblast monocultures to exclude the possibility of pneumonocyte influence on EGF-R in fetal fibroblasts. However, if the regulation of EGF-R in fibroblasts during fetal lung development depends on pneumonocytes, results from experiments in mixed lung cell cultures may differ from those obtained in monocultures.
The resulting changes in EGF-R binding in this study in response to treatment might have been secondary to alterations in receptor protein amounts, receptor internalization, and receptor degradation. Our Western blot analyses indicate that the receptor protein abundance changed in a similar manner to changes in binding after treatment with the different hormones. Similar correlations between changes in EGF-R protein and EGF-R binding have been shown in other systems for RA (48), EGF (57), and cortisol (58). The differences in the magnitude of response observed in binding studies compared with Western blot analyses may be the result of differences in the sensitivities of these two methods. EGF-R binding has been used to monitor changes in the differentiation of many fibroblast and epithelial cell types (36). The specificity of this intact cell method for measuring EGF-R specific binding has previously been shown using specific EGF-R antibodies (26).
In summary, we found that of the positive regulators in lung development, EGF and cortisol had little additional stimulatory effect on EGF-R binding at that time in gestation when the endogenous EGF-R binding in fibroblasts approaches its maximum as a sign of already acquired differentiation potential. RA markedly stimulated binding regardless of the gestational age. In contrast, the negative regulators DHT and TGF-ß1 both stimulated EGF-R binding, albeit with different intensities at different time-points. The responses to treatment were sex specific. It might be speculated that substances that are usually ascribed a certain role in EGF-R modulation, i.e. inhibitors or stimulators of EGF-R binding, switch between these functions according to the developmental status of the cell, i.e. early suppression, late stimulation, or vice versa. This hypothesis would assume the existence of an internal clock or zeitgeber regulating fibroblast maturation. EGF and its receptor might play a central role in the regulation of such a zeitgeber. Further studies are necessary to clarify the role of the EGF-R in regulating cell growth and cell maturation at this particular time in gestation.
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Footnotes |
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Received August 18, 1997.
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