Division of Newborn Medicine, Tufts-New England Medical Center, Boston, Massachusetts 02111
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ABSTRACT |
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Epidermal growth factor (EGF)
receptor (EGFR) regulates development of cell-cell communication in
fetal lung, but the signal transduction mechanisms involved are
unknown. We hypothesized that, in late-gestation fetal rat lung,
phospholipase C- (PLC-
) expression and activation by EGF is cell
specific and developmentally regulated. PLC-
immunolocalized to
cuboidal epithelium and mesenchymal clusters underlying
developing saccules. PLC-
protein increased from day
17 to day 19 and then decreased. In cultured fetal lung fibroblasts, EGF stimulated PLC-
phosphorylation 2.6-fold (day 17), 10.8-fold (day 19), and 4.2-fold (day
21). EGF stimulated 3H-labeled diacylglycerol
production in fibroblasts (beginning on day 18 in female and
on day 19 in male rats), but not in type II cells at any
time during gestation. EGFR blockade abrogated the observed stimulation
of PLC-
phosphorylation by EGF. In conclusion, PLC-
expression
and activation by EGF in fetal lung are cell specific, corresponding to
the development of EGFR expression. EGF induces diacylglycerol
production in a cell- and gestation-specific manner. PLC-
activation
by EGFR in fetal lung fibroblasts may be involved in EGF control of
lung development.
epidermal growth factor receptor; fibroblast-type II cell communication
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INTRODUCTION |
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DEVELOPMENT OF FIBROBLAST-TYPE II cell communication leading to fetal lung surfactant production is vital in preparing the fetus for extrauterine life. Previous studies have shown that this communication occurs during a specific gestational window that occurs earlier in females (16). Several hormones and growth factors regulate development of this fibroblast-type II cell communication. In the fetal rat and mouse lung fibroblast, epidermal growth factor (EGF), a 6-kDa growth factor, upregulates and advances development of fibroblast-type II cell communication (16, 23). This is the mechanism by which EGF stimulates fetal lung disaturated phosphatidylcholine (DSPC) production (9) and promotes surfactant-associated protein synthesis (17, 30). EGF acts by binding to a specific receptor, the EGF receptor (EGFR), leading to receptor dimerization and tyrosine phosphorylation, which results in receptor activation (1, 10). EGFR phosphorylation causes activation of specific intracellular proteins involved in EGFR signal transduction pathways, ultimately controlling gene expression.
We previously showed that EGFR expression and activation in fetal lung maturation are cell and gender specific, peaking earlier in females than in males, and correspond to the onset of fibroblast-type II cell communication, stimulating fetal lung surfactant synthesis (21). Immature fetal rat lung fibroblasts transduced with wild-type EGFR stimulated type II cell DSPC synthesis (20).
In general, EGFR activation may stimulate a variety of responses,
including proliferation, cell motility, and cell differentiation. These
responses can be ligand specific (e.g., EGF vs. transforming growth
factor-) and are specified in part through ligand discrimination and
differential receptor phosphorylation of specific intracellular second
messengers (3, 25). Thus EGF and transforming growth factor-
bind to and activate the EGFR but may activate different signaling pathways. The specific EGFR signal transduction
pathway that controls development of fibroblast-type II cell
communication leading to surfactant production in fetal lung is unknown.
One of the major signal transduction pathways activated by the EGFR
involves binding of the enzyme phospholipase C (PLC)- to the
receptor at the Src homology 2 sites, leading to phosphorylation and
activation of PLC-
. The EGFR-induced membrane association and
phosphorylation of PLC-
stimulate phosphoinositide breakdown by
increasing association of the enzyme with its substrate
(27). Phosphorylated PLC-
hydrolyzes
phosphatidylinositol 4,5-bisphosphate to diacylglycerol (DAG), which
activates protein kinase C, and inositol 1,4,5-trisphosphate, which
mobilizes Ca2+ from intracellular stores. PLC-
activation is often associated with cell differentiation
(12). To better understand the EGFR control of
fibroblast-type II cell communication, it is first important to
demonstrate that PLC-
is expressed and activated by EGF in fetal
lung fibroblasts during lung maturation.
We hypothesized that, in the fetal lung during development of
fibroblast-type II cell communication, EGF stimulates PLC- expression and activation in a cell-specific and developmentally regulated manner. We further hypothesized that EGF will stimulate DAG
production in a development-specific manner reminiscent of the temporal
development of cell-cell communication leading to surfactant synthesis.
To test this hypothesis, we studied the developmental expression and
cell-specific activation of PLC-
by EGF in fetal rat lung using
immunohistochemistry and Western blot and the developmental pattern of
DAG production in response to EGF stimulation in primary cultures of
fetal rat lung fibroblasts and type II cells.
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MATERIALS AND METHODS |
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Timed-pregnant Sprague-Dawley rats were purchased from Taconic
Farms (Germantown, NY). The following reagents were purchased as
indicated: Dulbecco's minimal essential medium (DMEM), Hanks' balanced salt solution, trypsin, and culture dishes from Life Technologies (Grand Island, NY); collagenase from Worthington Biochemical (Freehold, NJ); fetal calf serum (FCS) from Hyclone Laboratories (Logan, UT); gelatin sponges (Gelfoam) from Upjohn (Kalamazoo, MI); and OCT from Miles Laboratories (Elkhardt, IN). Proteinase and phosphatase inhibitors (aprotinin, leupeptin, pepstatin, and phenylmethylsulfonyl fluoride), mouse EGF (receptor grade), HEPES,
Triton X-100, Ca2+ ionophore A-23187, and tyrphostin
AG-1478 were purchased from Sigma (St. Louis, MO); anti-mouse IgG
antibody linked to horseradish peroxidase from NEN Life Science
Products (Boston, MA); protein A-Sepharose CL-4B from Amersham
Pharmacia Biotech (Piscataway, NJ); rabbit polyclonal antibody against
PLC-1 and EGFR blocking antibody (Ab 528) from Santa Cruz
Biotechnology (Santa Cruz, CA); and recombinant horseradish
peroxidase-linked antiphosphotyrosine antibody RC 20 from Transduction
Laboratories (Lexington, KY).
The institutional animal research committee approved the animal
research protocol. Pregnant rats at gestational days
17-21 (full term = 22 days) were killed by
CO2 inhalation. Fetuses were removed by hysterotomy, fetal
sex was identified, and fetal lungs were removed. In some experiments,
lungs from each gestational age and sex were fixed in 4%
paraformaldehyde and mounted in OCT for immunostaining or minced in
lysis buffer containing protease and phosphatase inhibitors to study
whole lung PLC- expression. In other experiments, lungs were pooled
by sex, and primary fetal lung fibroblast and type II cell cultures
were prepared to study cell- and gestation-specific PLC-
expression
and activation by EGF as well as stimulation of DAG production by EGF.
Whole lung PLC- immunostaining.
PLC-
expression was studied in whole lung sections from days
17, 19, and 21 of rat gestation, as described elsewhere
(21). Briefly, lungs were fixed in 4% paraformaldehyde
for 4 h, immersed overnight in 30% sucrose at 4°C, embedded in
OCT, and cut into 8-µm sections in a cryostat at
24°C. Serial
sections were then mounted onto room-temperature slides coated with
0.1% chromium persulfate-1% gelatin (Aldrich, Milwaukee, WI). Mounted
sections were washed for 20 min in 10 mM
Tris · HCl, 150 mM NaCl, and 0.05% Tween 20, pH
7.5. All subsequent washes were done in Tris-buffered saline-Tween 20 for 10 min. Nonspecific binding sites were blocked by sequential
incubations with avidin (15 min), biotin (15 min), and 1.5% normal
goat serum (20 min). Experimental sections were then incubated with a
rabbit polyclonal PLC-
IgG antibody (1:800), and control sections
were incubated with preimmune rabbit serum overnight at 4°C. On the
following day, sections were sequentially incubated at room temperature
with goat anti-rabbit biotinylated IgG antibody (1:1,000 dilution) for
60 min, avidin-biotin complex conjugated to alkaline phosphatase for 30 min, and finally alkaline phosphatase chromogen solution for 30 min
(Vectastain ABC-AP kit). The alkaline phosphatase reaction was stopped
by washing in distilled H2O. Sections were then
counterstained with nuclear fast red and dehydrated through a graded
alcohol series, coverslips were applied, and the sections were analyzed
by light microscopy.
Primary fetal lung fibroblast cultures. The fibroblasts were cultured using the process of differential adherence, as described previously by our laboratory (16, 21). Briefly, fetal lungs were pooled according to sex and minced using a razor blade. The minced lungs were dissociated in Hanks' balanced salt solution containing DNase and trypsin in a 37°C water bath for 10 min. The reaction was stopped using DMEM containing 10% stripped FCS, and a single-cell suspension was obtained by filtering through a sterile 70-µm cell strainer and centrifugation at 650 g for 10 min at 4°C. The pellet was resuspended in DMEM containing 10% stripped FCS and plated in 100-mm culture dishes (immunoprecipitation) or six-well plates (EGFR blocking experiments and DAG assays) and grown to confluence in DMEM containing 10% stripped FCS. At 85-90% confluence, the medium was removed and replaced with serum- and EGF-free medium and further incubated for 16-24 h. Serum-free medium was added for the same duration to the control cells. At the end of this period, cells were stimulated with EGF or used as control and processed for immunoprecipitation experiments or experiments to study stimulation of DAG production by EGF, as described below.
Alveolar type II cells. Fetal type II cells were isolated and cultured as previously described (16, 21). Briefly, at the end of the first differential adherence step described above, the culture supernatant was collected and centrifuged at 1,500 rpm at 4°C for 5 min. The pellet was resuspended in a smaller amount of DMEM + 10% stripped FCS to create a concentrated cell suspension for plating onto small pieces of sterile absorbable gelatin sponges, which had been presoaked in DMEM + 10% FCS (50 µl of cell suspension per 1 cm2 of Gelfoam). Gelatin sponges containing concentrated cell suspensions were placed onto petri dishes containing small amounts of DMEM + 10% stripped FCS and incubated overnight at 37°C. The gelatin sponges were then immersed in DMEM + 0.10% collagenase (200 units of activity per milligram), heated at 37°C for 3.5 h to allow enzymatic digestion of the gelatin sponges, and then placed in a 70-ml flask for a second differential adherence step for 1 h. The type II cells in the supernatant were centrifuged, resuspended in DMEM + 10% FCS, and plated at 0.5 × 106 cells per well in six-well plates. We previously showed that the purity of the type II cell cultures exceeded 90% (21). The type II cells were grown to confluence in DMEM + 10% stripped FCS.
EGFR-blocking experiments. Confluent cultures of fibroblasts in six-well plates were serum starved for 24 h. Some of the wells were used as controls, and others were treated with tyrphostin (0.05, 0.1, 0.5, 2.5, and 10 µM) to block EGFR phosphorylation (18) or with an EGFR-blocking antibody (0.5, 1, 2.5, 5, and 10 µg/ml) to block EGFR activation by EGF for 14 h. Half of the treated cultures were then stimulated with EGF (100 ng/ml for 3 min at 37°C) before the cells were harvested, and the other half of the cultures were harvested as unstimulated controls for Western blot experiments.
Immunoprecipitation and Western blot.
Immunoprecipitation and Western blot were carried out as described
elsewhere (6, 7, 21). Briefly, cells were stimulated with
EGF (100 ng/ml for 3 min at 37°C), scraped in lysis buffer (20 mM
HEPES, pH 7.4, 1 mM EDTA, 150 mM NaCl, and 1% Nonidet P-40) containing
protease and phosphatase inhibitors (phenylmethylsulfonyl fluoride,
leupeptin, pepstatin, aprotinin, vanadate, NaF, and -glycerophosphate), and centrifuged. The supernatant was collected, and protein content was measured using the Lowry protein assay (14). Anti-PLC-
antibody (2 µg) was added to 200 µg
of total lung protein or 200 µg of protein from fetal lung
fibroblasts and incubated at 4°C for 1.5 h with shaking. Protein
A-Sepharose (40 µl) was added, and the incubation was continued at
4°C for another 1.5 h with shaking followed by centrifugation.
The pellet was washed in washing buffer containing protease and
phosphatase inhibitors. The samples were mixed with SDS lysis buffer,
electrophoresed on an SDS-7.5% polyacrylamide separating gel, and
transferred semidry to a polyvinylidene difluoride membrane. Equal
loading was confirmed by Ponceau staining. After blocking for
nonspecific binding (1% BSA for antiphosphotyrosine antibody and 5%
milk for anti-PLC-
antibody) for 2 h at room temperature,
Western blots were probed with antiphosphotyrosine antibody, and the
bands were visualized using a horseradish peroxidase-linked
chemiluminescence kit and quantitated by densitometry. The blots were
then stripped, blocked, and reprobed with anti-PLC-
antibody, and
the bands were visualized by chemiluminescence and quantitated by
densitometry. Data are expressed as percentage of control.
Stimulation of DAG production. Preliminary studies performed to identify the optimal time and dose of EGF stimulation required for DAG production demonstrated that the minimal incubation condition for maximal DAG production was 5 ng/ml of EGF for 10 min. Confluent cultures (80%) of sex-specific fibroblasts and alveolar type II cells were washed with DMEM and loaded with [3H]glycerol (4 µCi/well) by incubation for 48 h to allow for radioactive label saturation for phosphoinositides. After 48 h, the medium was aspirated and fresh DMEM containing 10% stripped FCS was added. Cells in three wells on each six-well plate were incubated with unlabeled EGF (5 ng/ml) for 10 min, and then the EGF-containing medium was removed. The remaining three wells were left untreated as controls. Cells were then harvested in PBS, and aliquots were removed for protein assay using the method of Lowry et al. (14). DAG was isolated by lipid extraction followed by thin-layer chromatography (TLC) according to the method of Corey and Rosoff (5). The pellets were extracted in ice-cold 1:1:1 CHCl3-CH3OH-1 N HCl. The organic phase was collected, dried under N2, and resuspended in 50 µl of CHCl3. Twenty microliters of each sample were applied to Silica Gel 60 TLC plates (Merck) and chromatographed in 7:3 benzene-ethyl acetate. The plates were sprayed with H2SO4 and heated to 180°C to expose the spots. Spots corresponding to DAG standard were scraped into scintillation fluid, and disintegrations per minute (dpm) were measured using a scintillation counter (5, 22).
In some experiments, we used the Ca2+ ionophore A-23187 to stimulate DAG production through a non-tyrosine kinase receptor mechanism. Cultures of primary fetal lung fibroblasts and fetal type II cells were prepared and loaded with [3H]glycerol as described above and then washed, and the experimental cells were exposed to 10Data analysis.
Data were analyzed using the Mann-Whitney U test (PLC-
experiments) and t-test (DAG experiments). Values were
considered significant at P < 0.05.
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RESULTS |
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Immunostaining was used to determine the location of PLC-
expression in fetal lung and learn whether cell-specific changes occurred with fetal development. Figure 1
demonstrates PLC-
immunolocalization in fetal rat lung on days
17, 19, and 21. Throughout the gestational period studied, PLC-
immunostaining was present in the cuboidal epithelium of terminal airways, in mesenchyme, and rarely in columnar epithelium. Cuboidal epithelial staining was localized to the luminal
surface on day 17 but was distributed throughout the
cytoplasm on days 19 and 21. Mesenchymal staining
was diffuse on day 17 and became progressively localized to
clusters of fibroblasts adjacent to the developing saccules on
days 19 and 21.
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Quantitative changes in PLC- expression and in vivo tyrosine
phosphorylation in male and female developing rat lung were evaluated
using immunoprecipitation and Western blot techniques. Figure
2 shows a representative immunoblot and
densitometry of day 17, 19, and 21 fetal rat lung
protein immunoprecipitated and immunoprobed with an anti-PLC-
antibody. PLC-
protein was seen as an ~150-kDa band. Changes in
PLC-
expression with gestation appeared similar in male and female
rats; therefore, male and female results were combined for analysis.
Baseline PLC-
expression increased twofold from day 17 to
day 19 of gestation and then decreased from day
19 to day 21 (0.72 ± 0.08, 1.52 ± 0.27, and 0.78 ± 0.08 days 17, 19, and 21,
respectively, P < 0.05). Although the decrease in
PLC-
protein content from day 19 to day 21 appeared to be more pronounced in the female rat, statistical analysis of multiple blots did not demonstrate a significant difference between
male and female rats (37% decrease in male and 42% in female compared
with respective day 19 expression, n = 6).
This decrease between day 19 and day 21 is
consistent with the appearance on immunocytochemistry. Densitometry
results of phosphorylated PLC-
in whole lung are shown in Fig.
2C. The data were normalized to day 17 within
each blot. Again, gestational changes appeared similar in male and
female rats, and these were combined for analysis. The level of in vivo
PLC-
phosphorylation was minimal on day 17 (relative
densitometry = 1.0 ± 0.15). There was a 1.5-fold increase in
phosphorylated PLC-
on day 19 (1.5 ± 0.075, P < 0.05, n = 6) followed by a sharp
decrease on day 21 (0.9 ± 0.22). In each experiment,
the ratio of phosphorylated to total PLC-
increased with gestation;
however, the magnitude of the increase could not be quantified among
experiments because of different exposure times of blots in different
experiments.
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To further understand the cell- and development-specific regulation of
PLC- activation by EGF and gain insight into its potential activity
in fibroblast-type II cell communication, we studied PLC-
expression
using primary cultures of fetal rat lung fibroblasts from days
17, 19, and 21 of gestation. PLC-
protein was
expressed and was stable with time in culture in control and
EGF-stimulated fetal lung fibroblasts on all the days studied. A
representative immunoblot and densitometry from all blots are shown in
Fig. 3A. We then studied
PLC-
phosphorylation in response to EGF stimulation. A
representative Western blot of protein from female fetal lung fibroblasts immunoprecipitated with anti-PLC-
antibody and
immunoprobed with an antiphosphotyrosine antibody and densitometry
results from all blots are shown in Fig. 3B. PLC-
phosphorylation was minimal in control fibroblasts. EGF significantly
stimulated PLC-
phosphorylation in a development-specific pattern.
In day 17 fibroblasts, PLC-
phosphorylation was 2.6-fold
higher in EGF-stimulated cells than in controls, but this was not
statistically significant. On day 19, EGF induced a more
profound (10.8-fold) stimulation in PLC-
phosphorylation than in
controls (P = 0.002). This relative degree of
stimulation was also significantly greater than that on day
17 with EGF stimulation (P = 0.01). Comparison of
male and female rats indicated that this magnitude of stimulation was greater in the male rats (data not shown). On day 21, EGF
also induced a significant (4.2-fold) increase in PLC-
phosphorylation (P = 0.01), but this was not as
profound as on day 19.
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To further understand whether the gestational changes in PLC-
phosphorylation by EGF were mediated via the EGFR, we used tyrphostin
to block EGFR phosphorylation and an EGFR-blocking antibody to block
EGFR activation by EGF. These results are shown in Fig.
4. Dose-response studies showed maximal
inhibition of EGFR activation with 10 µM tyrphostin and with
EGFR-blocking antibody at 10 µg/ml. Blocking the activity of EGF on
the EGFR and blocking EGFR phosphorylation in response to EGF each
abrogated stimulation of EGFR phosphorylation and PLC-
activation by
EGF.
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Inasmuch as one of the signal transduction intermediates created by
PLC- activation in its signal transduction pathway is DAG, we
studied cell-specific DAG production in response to EGFR activation and
non-tyrosine kinase receptor mechanisms. Figure 5 shows DAG production in response to
EGFR activation in male and female fetal rat lung fibroblasts. Control
fibroblasts exhibited a basal level of [3H]DAG production
that did not change with gestation in male or female rats. However, DAG
production in response to EGFR activation showed a sex-specific
developmental pattern. In male fibroblasts, EGF did not induce
stimulation of [3H]DAG production on day 17 or
day 18 but did induce a 2.3-fold increase in DAG production
on gestational day 19 [1,671 ± 340 and 3,946 ± 550 (SD) dpm/mg protein for control and EGF-treated fibroblasts,
respectively, P = 0.003]. This effect persisted on day 21 (2,057 ± 985 and 4,675 ± 272 dpm/mg
protein for control and EGF-treated fibroblasts, respectively,
P = 0.01). In the female rat, EGF caused no stimulation
of [3H]DAG production on day 17, similar to
the results from the male rat on day 17. However, in
day 18 female fibroblasts, EGF induced a 2.2-fold increase
in DAG production (1,579 ± 114 and 3,481 ± 270 dpm/mg
protein for control and EGF-treated fibroblasts, respectively, P = 0.004). This response persisted on day
19 (2,116 ± 225 and 3,977 ± 1,167 dpm/mg protein for
control and EGF-treated fibroblasts, respectively, P < 0.05) and day 21 (2,024 ± 765 and 4,024 ± 866 dpm/mg protein for control and EGF-treated fibroblasts, respectively, P = 0.04).
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The effect of EGFR activation on DAG production in male and female
alveolar type II cells is shown in Fig.
6. There were no changes in the basal
levels of DAG in control cells from day 17 through day
21, and these baseline levels were similar to those in untreated
fibroblasts. In contrast to the effect of EGF on fibroblasts, EGF did
not have an effect on DAG production at any gestational age in male or
female rats.
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To study the EGFR-independent development of PLC- production of DAG
in fetal lung, we studied the production of DAG after stimulation with
the Ca2+ ionophore A-23187 and compared this with
EGF-induced DAG production in fetal rat lung fibroblasts and type II
cells. Baseline unstimulated DAG production was similar to that shown
in Figs. 4 and 5. A-23187 treatment did not stimulate DAG production in
day 17 fibroblasts. However, on day 21, there was
a 2.2-fold increase in [3H]DAG production in fibroblasts
exposed to the Ca2+ ionophore. The positive control
fibroblasts exhibited the same response to EGF as in the previous
experiments (2-fold increase in DAG production compared with controls).
Similar responses were seen in male fibroblasts (3.7-fold increase on
day 21). Interestingly, the DAG response of alveolar type II
cells to Ca2+ ionophore stimulation was not the same as the
response to EGF. As expected, there was no response to A-23187 on
day 17 in male or female rats. However, on day
21, A-23187 induced a 2.8-fold increase in male type II cells and
a 2.2-fold increase in [3H]DAG production in female type
II cells. Consistent with our previous results, the positive control
(EGF-stimulated) type II cells showed no stimulation of DAG production.
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DISCUSSION |
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There is growing evidence that EGF and EGFR have a primary role in
normal lung development (4, 8, 11, 19, 31). The capacity
of EGF to modulate cell growth and differentiation is mediated through
EGFR activation, which generates an intracellular biochemical signaling
cascade. These cellular responses can be ligand specific, causing
differential receptor phosphorylation and activation by the receptor of
specific intracellular second messengers (25). The
specific EGFR signal transduction pathway(s) involved in the
development of fibroblast-type II cell communication is unknown. One of
the major EGFR signal transduction pathways involved in cell
differentiation involves phosphorylation of PLC-. Gene-targeting and
disruption experiments have shown that PLC-
is essential for
embryonic development during early gestation, inasmuch as
PLC-
/
embryos die before embryonic day 9.
These embryos were unable to mobilize intracellular Ca2+
(13). The present experiments were undertaken to determine whether PLC-
is expressed and activated in developing fetal lung in
a manner consistent with a potential role in mediating fibroblast-type II cell communication.
Immunocytochemistry of fetal rat and mouse lung showed that EGFR is
strongly localized to fibroblast clusters underlying epithelial cells
of airways and alveoli as well as scattered columnar epithelial cells
(21, 28). A transgenic mouse model in which the EGFR gene
was replaced with a -galactosidase gene driven by the EGFR promoter
also demonstrated that EGFR expression in fetal lung is primarily found
in fibroblasts adjacent to airway and alveolar epithelium
(24). At birth, EGFR knockout mice have immature lungs,
characterized by decreased air spaces, increased interstitial tissues,
and increased fetal mortality in the homozygotes (15, 26,
28). Immature fetal rat lung fibroblasts transduced with wild-type EGFR stimulated type II cell DSPC synthesis
(20), and EGFR autoactivation in the lung has been
described (29).
Using immunocytochemistry, we found that PLC- became more localized
to the subepithelial fibroblasts during the progression of late fetal
rat lung development, similar to the spatial and temporal expression of
the EGFR during the same interval of lung development. Furthermore,
developmental changes in PLC-
protein indicated by the
immunostaining were confirmed by Western blot of whole lung, which
showed that PLC-
expression was maximal on day 19 and
decreased on day 21. This is also consistent with the timing
of the development of EGFR in fetal lung (21). In contrast
to studies of EGFR development, we did not observe a sex-specific
difference in the development of PLC-
expression. There are several
possibilities for this apparent lack of sex difference. Sex differences
in EGF responsiveness during development of fibroblast-type II cell
communication may arise at the level of the receptor, rather than this
signal transduction pathway. It is also possible that a sex difference
in the development of PLC-
expression occurs but within a more
narrow window than the time points we studied. Alternatively, the lack
of an observed sex difference may be due to the evaluation of the whole
lung, whereas sex-specific developmental differences in timing of
expression may be confined to just one cell type, such as the fibroblast.
Multiple studies show that fetal lung fibroblasts in primary culture
exhibit a developmental phenotype in cell-cell communication and EGFR
expression (2, 4, 16, 21). The development of EGFR
expression and activity in late-gestation fetal lung fibroblasts is sex
and gestation specific, peaking in the fetal rat lung on day
18 in the female and on day 19 in the male. This timing
corresponds to the onset of fibroblast-type II cell communication
controlling type II cell maturation and surfactant synthesis. Fetal
type II cells, in contrast, demonstrate very low EGF binding and EGFR protein expression with no change during development (21).
Our data demonstrate that PLC- protein expression remained stable in
fetal lung fibroblasts across the gestational days studied, even though
the whole lung PLC-
expression showed a specific developmental
pattern. This difference may possibly be due to the presence or absence
of specific mediators that occur in vivo. However, EGF-induced PLC-
phosphorylation in fetal lung fibroblasts is maximal on day
19 when EGFR expression is maximal. These data indicate that
PLC-
protein is present at the appropriate location and time of
gestation in the developing lung to be involved in the EGFR signal
transduction pathway controlling the development of fibroblast-type II
cell communication.
One of the strategies by which signals are transduced from the cell
membrane receptors to intracellular processes is the generation of
intracellular second messengers that interact with specific intracellular proteins to trigger a specific cellular response (3). PLC- phosphorylation by the EGFR results in
generation of the intracellular second messengers Ca2+ and
DAG. Using primary cultures of fetal rat lung fibroblasts and type II
cells, we have shown that DAG production was stimulated by EGF in
primary fetal lung fibroblasts but not in type II cells. This effect
first appeared on gestational day 18 in the females and
day 19 in the males, corresponding to the time when
fibroblasts exhibit a sharp increase in EGFR amount (21)
as well as maximal PLC-
expression. This indicates that stimulation
of DAG via EGFR signaling occurs in a cell- and sex-specific manner
that corresponds to the developmental expression of EGFR-binding activity.
We observed no sex-specific differences in PLC- expression and
activation by EGF (on days 17, 19, and 21).
However, we observed a significant difference in stimulation of DAG
production by EGF (day 18). It is possible that there may be
sex-specific differences in PLC-
activation by EGF at this time
point in gestation, inasmuch as we previously showed that EGFR
expression is sex specific in the fetal lung fibroblast, with the
primary difference in fetal rat occurring on day 18.
Inasmuch as the primary focus of this study was to demonstrate the
cell- and development-specific regulation of PLC-
activation by EGF,
we did not evaluate all possible developmental time points.
We wanted to investigate whether the developmental differences in
stimulation of DAG by EGF were solely through EGF-induced EGFR
activation. DAG may be produced from sources other than the action of
PLC- (phospholipase D, phosphatidic acid phosphohydrolase, and de
novo biosynthesis). We used the Ca2+ ionophore
A-23187 to stimulate DAG production through a non-tyrosine kinase
receptor mechanism and found interesting differences from the EGF
effect. A-23187 did not stimulate DAG production in primary fetal lung
fibroblasts or alveolar type II cells on gestational day 17.
However, there was a strong stimulation of DAG by A-23187 in
fibroblasts and type II cells on day 21. This contrasts with the developmental pattern of DAG production in response to EGF. These
data suggest that day specificity and cell specificity are regulated
differently and involve tyrosine kinase- and non-tyrosine kinase-modulated pathways. Development of DAG production in
the fetal lung may be regulated by EGFR-dependent as well as
EGFR-independent mechanisms. We speculate that, in type II cell
development, PLC-
may be upregulated by mechanisms other than EGF stimulation.
In summary, our data indicate that PLC- is expressed and activated
by EGF in fetal lung in a cell-specific manner that corresponds to the
developmental expression of the EGFR. In addition, we have shown that
EGF induces the intracellular second messenger DAG in a cell- and
gestation-specific manner. We speculate that this cell-specific PLC-
phosphorylation by EGFR during fetal lung maturation initiates specific
signal transduction events, such as generation of the intracellular
second messenger DAG, to control the development of fibroblast-type II
cell communication leading to type II cell maturation and surfactant
synthesis. Further experiments to demonstrate the functional importance
of this EGFR signaling pathway in controlling lung maturation are vital
to understand the mechanisms controlling fetal lung maturation.
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ACKNOWLEDGEMENTS |
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This study was supported by American Lung Association Grant RG-043N, National Heart, Lung, and Blood Institute Grant HL-37930, and the Peabody Foundation.
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FOOTNOTES |
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Address for reprint requests and other correspondence: S. M Ramadurai, Div. of Newborn Medicine, Tufts-New England Medical Center, Box 44, 750 Washington St., Boston, MA 02111 (E-mail: sramadurai{at}tufts-nemc.org).
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.
First published December 27, 2002;10.1152/ajplung.00117.2002
Received 19 April 2002; accepted in final form 18 December 2002.
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