Cell-specific and developmental expression of phospholipase C-gamma and diacylglycerol in fetal lung

Sujatha M. Ramadurai, Wu-Yuan Chen, George B. Yerozolimsky, Michelle Zagami, Christiane E. L. Dammann, and Heber C. Nielsen

Division of Newborn Medicine, Tufts-New England Medical Center, Boston, Massachusetts 02111


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-gamma (PLC-gamma ) expression and activation by EGF is cell specific and developmentally regulated. PLC-gamma immunolocalized to cuboidal epithelium and mesenchymal clusters underlying developing saccules. PLC-gamma protein increased from day 17 to day 19 and then decreased. In cultured fetal lung fibroblasts, EGF stimulated PLC-gamma 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-gamma phosphorylation by EGF. In conclusion, PLC-gamma 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-gamma 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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-alpha ) 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-alpha 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)-gamma to the receptor at the Src homology 2 sites, leading to phosphorylation and activation of PLC-gamma . The EGFR-induced membrane association and phosphorylation of PLC-gamma stimulate phosphoinositide breakdown by increasing association of the enzyme with its substrate (27). Phosphorylated PLC-gamma 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-gamma 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-gamma 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-gamma 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-gamma 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.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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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-gamma 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-gamma 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-gamma expression and activation by EGF as well as stimulation of DAG production by EGF.

Whole lung PLC-gamma immunostaining. PLC-gamma 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-gamma 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 beta -glycerophosphate), and centrifuged. The supernatant was collected, and protein content was measured using the Lowry protein assay (14). Anti-PLC-gamma 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-gamma 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-gamma 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 10-6 M A-23187 for 10 min to stimulate [3H]DAG production through the PLC-gamma intracellular pathway, bypassing the EGFR. Additionally, in each experiment, some cells were stimulated with EGF for 10 min to stimulate EGFR activation and subsequent [3H]DAG production. Control cells received no stimulation. Cells were harvested, and [3H]DAG was isolated by TLC and measured by scintillation counter (see above).

Data analysis. Data were analyzed using the Mann-Whitney U test (PLC-gamma experiments) and t-test (DAG experiments). Values were considered significant at P < 0.05.


    RESULTS
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ABSTRACT
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RESULTS
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REFERENCES

Immunostaining was used to determine the location of PLC-gamma expression in fetal lung and learn whether cell-specific changes occurred with fetal development. Figure 1 demonstrates PLC-gamma immunolocalization in fetal rat lung on days 17, 19, and 21. Throughout the gestational period studied, PLC-gamma 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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Fig. 1.   Phospholipase C-gamma (PLC-gamma ) immunostaining in day 17 (A), day 19 (B), and day 21 (C) male fetal rat lung. Cuboidal epithelial staining (arrowheads) was localized to luminal surface on day 17 and throughout cytoplasm on days 19 and 21. Mesenchymal staining (arrows) was diffuse on day 17 and became progressively localized to clusters of fibroblasts adjacent to developing saccules on days 19 and 21. Magnification ×400.

Quantitative changes in PLC-gamma 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-gamma antibody. PLC-gamma protein was seen as an ~150-kDa band. Changes in PLC-gamma expression with gestation appeared similar in male and female rats; therefore, male and female results were combined for analysis. Baseline PLC-gamma 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-gamma 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-gamma 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-gamma phosphorylation was minimal on day 17 (relative densitometry = 1.0 ± 0.15). There was a 1.5-fold increase in phosphorylated PLC-gamma 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-gamma 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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Fig. 2.   Representative immunoblot (A) and densitometry (B) of PLC-gamma expression and densitometry of tyrosine-phosphorylated PLC-gamma (C) in day 17, 19, and 21 male and female fetal rat lung. Total lung protein (200 µg) was immunoprecipitated (IP) and immunoprobed with an anti-PLC-gamma antibody (to study expression) or with an antiphosphotyrosine antibody. PLC-gamma protein was seen as an ~150-kDa band. Arrowhead, PLC-gamma . Results for males and females are combined (n = 6). Values are means ± SE. WB, Western blot. *P = 0.002 (B) and *P < 0.05 (C).

To further understand the cell- and development-specific regulation of PLC-gamma activation by EGF and gain insight into its potential activity in fibroblast-type II cell communication, we studied PLC-gamma expression using primary cultures of fetal rat lung fibroblasts from days 17, 19, and 21 of gestation. PLC-gamma 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-gamma phosphorylation in response to EGF stimulation. A representative Western blot of protein from female fetal lung fibroblasts immunoprecipitated with anti-PLC-gamma antibody and immunoprobed with an antiphosphotyrosine antibody and densitometry results from all blots are shown in Fig. 3B. PLC-gamma phosphorylation was minimal in control fibroblasts. EGF significantly stimulated PLC-gamma phosphorylation in a development-specific pattern. In day 17 fibroblasts, PLC-gamma 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-gamma 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-gamma phosphorylation (P = 0.01), but this was not as profound as on day 19.


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Fig. 3.   Representative immunoblot and densitometry of PLC-gamma expression (A) and PLC-gamma activation by epidermal growth factor (EGF, B) in day 17, 19, and 21 female fetal rat lung fibroblasts. Cells in EGF group were treated with EGF (100 ng/ml) for 3 min at 37°C. Lysates were immunoprecipitated with an anti-PLC-gamma antibody and immunoprobed with an anti-PLC-gamma antibody (A) or an antiphosphotyrosine (alpha ptyr) antibody (B). Values are means ± SE; n = 6. C, control; E, EGF. *P = 0.002; **P = 0.01; # P = 0.01.

To further understand whether the gestational changes in PLC-gamma 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-gamma activation by EGF.


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Fig. 4.   Effect of EGF receptor (EGFR) blockade with EGFR-blocking antibody (EGFR Ab, 10 µg/ml) and tyrphostin (10 µM) in control (-) and EGF-stimulated (+) cells. Cells were pretreated with EGFR blocking antibody or tyrphostin for 14 h and then stimulated with EGF (100 ng/ml) for 3 min at 37°C. Western blots were immunoprobed with an antiphosphotyrosine antibody and then stripped and reprobed with an anti-EGFR antibody followed by an anti-PLC-gamma antibody.

Inasmuch as one of the signal transduction intermediates created by PLC-gamma 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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Fig. 5.   Diacylglycerol (DAG) production [disintegrations per minute (dpm)/mg protein] in control and in response to EGFR activation by EGF in male (A) and female (B) fetal lung fibroblasts (days 17-21). Values are means ± SD of an average of 3-6 observations from 3 experiments. *P < 0.01 (A) and *P < 0.05 (B).

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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Fig. 6.   DAG production in control and in response to EGFR activation by EGF in male (A) and female (B) fetal type II cells (days 17-21). Values are means ± SD of an average of 3-6 observations from 3 experiments.

To study the EGFR-independent development of PLC-gamma 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.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-gamma . Gene-targeting and disruption experiments have shown that PLC-gamma is essential for embryonic development during early gestation, inasmuch as PLC-gamma -/- embryos die before embryonic day 9. These embryos were unable to mobilize intracellular Ca2+ (13). The present experiments were undertaken to determine whether PLC-gamma 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 beta -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-gamma 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-gamma protein indicated by the immunostaining were confirmed by Western blot of whole lung, which showed that PLC-gamma 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-gamma 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-gamma 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-gamma protein expression remained stable in fetal lung fibroblasts across the gestational days studied, even though the whole lung PLC-gamma 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-gamma phosphorylation in fetal lung fibroblasts is maximal on day 19 when EGFR expression is maximal. These data indicate that PLC-gamma 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-gamma 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-gamma 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-gamma 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-gamma 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-gamma 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-gamma (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-gamma may be upregulated by mechanisms other than EGF stimulation.

In summary, our data indicate that PLC-gamma 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-gamma 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.


    ACKNOWLEDGEMENTS

This study was supported by American Lung Association Grant RG-043N, National Heart, Lung, and Blood Institute Grant HL-37930, and the Peabody Foundation.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Lung Cell Mol Physiol 284(5):L808-L816
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