Effect of tyrosine kinase inhibition on surfactant protein A gene expression during human lung development

Jonathan M. Klein, Louis J. Dewild, and Troy A. McCarthy

Department of Pediatrics, University of Iowa, Iowa City, Iowa 52242-1083

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Epidermal growth factor (EGF) stimulates surfactant protein (SP) A synthesis in human fetal lung explants. Ligand binding to the EGF receptor stimulates an intrinsic receptor tyrosine kinase with subsequent activation of second messengers. We hypothesized that inhibition of EGF-receptor tyrosine kinase activity would block SP-A expression in spontaneously differentiating cultured human fetal lung tissue. Midtrimester fetal lung explants were exposed for 4 days to genistein (a broad-range inhibitor of tyrosine kinases) and tyrphostin AG-1478 (a specific inhibitor of EGF-receptor tyrosine kinase). Genistein significantly decreased SP-A and SP-A mRNA levels without affecting either tissue viability or the morphological differentiation of alveolar type II cells. Tyrphostin AG-1478 also decreased SP-A content and SP-A mRNA levels in cultured fetal lung explants. Treatment with EGF could not overcome the inhibitory effects of either genistein or tyrphostin on SP-A; however, only tyrphostin inhibited EGF-receptor tyrosine phosphorylation. We conclude that specific inhibition of EGF-receptor tyrosine kinase with tyrphostin AG-1478 blocks the expression of SP-A during spontaneous differentiation of cultured human fetal lung tissue. Furthermore, exposure to genistein also decreases SP-A expression and blocks the effects of EGF in human fetal lung tissue without inhibiting EGF-receptor tyrosine phosphorylation. These findings support the importance of tyrosine kinase-dependent signal transduction pathways in the regulation of SP-A during human fetal lung development.

epidermal growth factor receptor; phosphorylation; genistein; tyrphostin

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

EPIDERMAL GROWTH FACTOR (EGF) is one of many polypeptide hormones that influence fetal lung development. EGF modulates its effects by binding with high affinity to a specific cell-surface receptor, a 170-kDa transmembrane glycosylated phosphoprotein with intrinsic tyrosine kinase activity (4) that is essential for EGF-receptor function (6). Transforming growth factor-alpha (TGF-alpha ), a small protein with 40% homology to EGF, also binds with high affinity to the EGF receptor (16). Ligand binding results in dimerization of the EGF receptor with rapid activation of tyrosine kinase activity, leading to tyrosine autophosphorylation (3) and the subsequent tyrosine phosphorylation of specific substrate proteins that function as second messengers in a signal transduction pathway through which mitogenesis and other cellular activities are regulated (33).

EGF accelerates fetal lung maturation both in vivo and in vitro. EGF increases alveolarization and decreases the severity of respiratory distress syndrome (31) in fetal lambs. In fetal rabbits, EGF improves pulmonary compliance (5) and increases the production of surfactant-associated phospholipids (13). In fetal rhesus monkeys, EGF stimulates surfactant protein (SP) A synthesis, enhances alveolar differentiation, and reduces the severity of respiratory distress syndrome (11). In vitro, EGF stimulates surfactant-associated phospholipid synthesis (12) and alveolar type II cell differentiation (10) in fetal rat lung and branching morphogenesis in fetal mouse lung (34).

The role of the EGF receptor and its ligands in human fetal lung development has yet to be clearly defined. EGF has been shown to stimulate SP-A synthesis in cultured human fetal lung explants (36). Ligands for the EGF receptor have been found in human fetal lung tissue. Immunoreactive TGF-alpha has been identified in epithelial cells lining prealveolar ducts (30) and in both tracheal and distal airway epithelia throughout gestation (23). Immunoreactive EGF has also been identified in fetal tracheobronchial and distal airway epithelium (23). After 24 wk of gestation, both EGF and TGF-alpha are detectable by immunostaining in alveolar type II cells (23). EGF-receptor protein and mRNA have been detected in alveolar epithelium from midtrimester human fetal lung explants undergoing spontaneous differentiation in vitro (17) and in alveolar type II cells after 24 wk of gestation (23). Furthermore, mRNA for both ligands has been detected in mesenchymal tissue from human fetal lung throughout gestation (23), suggesting a paracrine effect of EGF/TGF-alpha on alveolar type II cell differentiation. The presence of EGF receptor and its ligands in distal pulmonary epithelium during human fetal lung development suggests a regulatory role for EGF in type II cell differentiation. The issue of whether EGF receptors are necessary for alveolar type II cell differentiation or just associated with alveolar epithelium during this process needs to be addressed.

One strategy to address the issue of causality would be to block human EGF-receptor activity. In other species, inhibiting EGF-receptor tyrosine kinase activity has interfered with fetal lung development. Tyrphostin inhibits branching morphogenesis in mouse embryo lungs (34) and, along with genistein, decreases thymidine incorporation and cell proliferation in rabbit type II cells (7). In EGF-receptor knockout mice, the alveolar epithelium is undifferentiated and the lungs are inadequately inflated (25). Blocking the ligand for the EGF receptor has also interfered with fetal lung development. Branching morphogenesis in mouse embryo lungs is decreased when EGF expression is inhibited with antisense oligonucleotides (24), and alveolar type II cell differentiation is inhibited in fetal mice treated with anti-mouse EGF antiserum (37).

This study addresses whether SP-A expression is dependent on an intact EGF-receptor signal transduction cascade. We hypothesized that inhibition of EGF-receptor function by blocking tyrosine kinase activity would lead to decreased expression of human SP-A. To test our hypothesis, we measured levels of SP-A and SP-A mRNA in spontaneously differentiating human fetal lung explants cultured for 4 days with either genistein [a broad-range inhibitor of tyrosine kinases (1, 2, 21)] or tyrphostin AG-1478 [a highly specific inhibitor of EGF-receptor tyrosine kinase (20)].

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Organ culture. The effects of genistein (Upstate Biotechnology, Lake Placid, NY) and tyrphostin AG-1478 (Calbiochem, San Diego, CA) on levels of SP-A and SP-A mRNA were evaluated in cultured human fetal lung explants. Human fetal lung tissue was obtained under a protocol approved by the University of Iowa Human Subjects Review Committee. The explants were prepared from lung tissue obtained from midtrimester abortuses (18-21 wk) as previously described (27). The major airways were removed, and the distal lung tissue was minced into 1-mm3 pieces with a razor blade under sterile conditions. The minced tissue was placed on a piece of lens paper resting on a metal grid inside a 35-mm culture dish containing 1 ml of serum-free Waymouths MB 752/1 medium (GIBCO Laboratories, Grand Island, NY) with added penicillin G (100 U/ml), streptomycin (100 µg/ml), and amphotericin B (0.25 µg/ml). The explants were incubated at 37°C in a humidified atmosphere of 5% CO2-95% air for 4 days, with the media changed daily. Starting tissue (human fetal lung tissue before culture) and the harvested explants were either fixed in 2.5% glutaraldehyde or frozen in liquid nitrogen and stored at -70°C until subsequent analysis. Experiments were conducted with explants prepared from individual fetuses.

Genistein and tyrphostin AG-1478 were diluted in DMSO, with the final concentration of DMSO in the culture medium not exceeding 0.1%. Experiments were conducted at the following concentrations: 1.0, 5.0, and 10 µg/ml (3.7-37 µM) genistein and 9.5 µg/ml (30 µM) tyrphostin AG-1478. Additionally, the explants were cultured with 39 µM daidzein (an inactive analog of genistein; Calbiochem), 30 µM tyrphostin A1 (a negative control for tyrphostins; Calbiochem), and the vehicle (DMSO). A control without DMSO, genistein, or tyrphostin was prepared for all experiments and used to normalize data between experiments. Cytotoxicity to genistein was evaluated by measuring the release of lactate dehydrogenase (LDH; LDH assay, LDL-20 kit, Sigma) into the media at the highest concentration of genistein (10 µg/ml) used. Additionally, viability of the tissue in the presence of the same concentration of genistein was evaluated at the ultrastructural level with electron microscopy by leaving the explants exposed to genistein for a total of 4-7 days, a time period in which lamellar bodies become abundant in cultured human fetal lung (28). The effect of EGF treatment (10 ng/ml) on SP-A content in the presence or absence of either genistein (37 µM) or tyrphostin AG-1478 (30 µM) was also evaluated in explants cultured for 4 days.

Western blot immunoanalysis of SP-A. Starting tissue and harvested explants were homogenized in PBS with 200 µM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, leupeptin (8 µg/ml), soybean trypsin inhibitor (50 µg/ml), and 5 mM EDTA. Samples were centrifuged (600 g) for 5 min, and supernatant protein (75 µg/lane) was separated by electrophoresis on a 12.5% SDS-polyacrylamide gel, transferred to an Immobilon-P membrane (Millipore, Bedford, MA), and blocked as described (29). The membrane was incubated for 1 h at room temperature with guinea pig polyclonal anti-human SP-A antibodies (1:1,000 dilution; kindly supplied by Dr. J. Snyder, Dept. of Anatomy, Univ. of Iowa), rinsed with double-distilled water, then incubated with sheep anti-guinea pig IgG conjugated to alkaline phosphatase (1:2,000 dilution; Boehringer Mannheim, Indianapolis, IN) for 1 h at room temperature, and then washed as previously described (29). The immunoreactive SP-A bands were then detected by incubating the membrane at room temperature for 30 min in a solution containing 100 mM Tris (pH 9.5), 100 mM NaCl, 5 mM MgCl2, 5-bromo-4-chloro-3-indoyl phosphate (165 µg/ml), and nitro blue tetrazolium (330 µg/ml). The membrane was rinsed in distilled water, dried, and photographed. The relative amount of immunoreactive SP-A present in each sample was quantitated by densitometry (Radioanalytic and Visual Imaging System, Ambis, San Diego, CA). The densitometric data from each blot were normalized to the control condition, with the control value set equal to one for each experiment.

Northern blot analysis of SP-A mRNA. Total RNA was isolated by a single-step acid-phenol-chloroform extraction method (8). Ten micrograms of total RNA per condition were separated by gel electrophoresis (1.2% agarose), transferred by capillary action to a nylon membrane (S&S Nytran, Schleicher & Schuell, Keene, NH), baked 30 min, ultraviolet cross-linked (UV Stratalinker 1800, Stratagene, LaJolla, CA), and prehybridized as described (9).

Hybridization using an SP-A cDNA probe (kindly supplied by J. Whitsett, Dept. of Pediatrics, Univ. of Cincinnati) was performed to evaluate steady-state levels of SP-A mRNA. The cDNA probes were radiolabeled with [alpha -32P]deoxycytidine triphosphate using a random-priming kit (Amersham, Arlington Heights, IL). Hybridization was performed as described (9), and mRNA levels were quantitated from the membrane by a beta counter (Ambis Radioanalytic and Visual Imaging System). The reactive bands were visualized by autoradiography at -70°C.

To control for loading artifacts, the density of the 18S rRNA bands, using the Ambis Visual Imaging System, were measured from photographs of RNA gels stained with ethidium. The gels were photographed before the capillary transfer of RNA. The density of the bands on the Northern blots was then normalized to the density of their corresponding 18S rRNA bands. For the tyrphostin experiments, in which the nylon membranes were still available, the membranes were stripped and reprobed with a 32P-labeled human 18S ribosomal cDNA probe (American Type Culture Collection). The densitometric data obtained from the SP-A Northern blots, after controlling for loading, were then normalized to the control condition, with the control condition set equal to one for each experiment.

Immunoprecipitation and immunoblotting of phosphotyrosine proteins. Homogenates (500 µg of total protein) prepared from explants described in Western blot immunoanalysis of SP-A were incubated with rabbit polyclonal anti-phosphotyrosine antibodies (5 µg, UBI) for 2 h at room temperature with shaking. Protein A-agarose beads (12.5 µl, GIBCO BRL) were added, and the mixture was incubated at room temperature for 1 h with shaking. Immune complexes were pelleted by centrifugation; washed with 150 mM NaCl, 200 µM sodium orthovanadate, and 10 mM Tris · HCl (pH 7.4); and repelleted. The pellet was resuspended by boiling, and the proteins were separated on a 7% SDS-PAGE gel. The phosphotyrosine proteins were then transferred to an Immobilon-P membrane (Millipore). Nonspecific binding was blocked by an overnight incubation at 4°C with 3% nonfat dry milk in PBS. The membrane was then incubated for 3 h at room temperature with a mouse monoclonal anti-phosphotyrosine antibody (1:1,000 dilution, UBI), rinsed, and then incubated with goat anti-mouse IgG conjugated to alkaline phosphatase (1:2,000 dilution, UBI) for 1 h at room temperature. The membrane was rinsed, and the immunoreactive tyrosine-phosphorylated bands were detected by incubating the membrane at 25°C for 30 min in 100 mM Tris (pH 9.5), 100 mM NaCl, 5 mM MgCl2, 5-bromo-4-chloro-3-indoyl phosphate (165 µg/ml), and nitro blue tetrazolium (330 µg/ml). The membrane was rinsed in distilled water, dried, and photographed. The relative amount of immunoreactive tyrosine-phosphorylated protein visualized at 170,000 Da, which is the presumptive tyrosine-phosphorylated EGF receptor, was quantitated by densitometry as described in Western blot immunoanalysis of SP-A.

Statistical analysis. The effects of tyrosine kinase inhibitors on SP-A protein, SP-A mRNA, and tyrosine-phosphorylated EGF receptor were evaluated by ANOVA with either a Bonferroni correction (GraphPad InStat, GraphPad Software, San Diego, CA), Tukey's test (SigmaStat), or Newman-Keuls test (WinStar) where appropriate for multiple comparisons. In experiments not involving multiple comparisons, the unpaired Student's t-test was used.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Effect of genistein on SP-A content. We measured levels of immunoreactive SP-A isolated from cultured human fetal lung explants that had been exposed to genistein for 4 days. The immunoblot analysis detected a 35-kDa band, consistent with the human pulmonary surfactant-associated glycoprotein (SP-A) that migrates from 34 to 36 kDa (35). The dose-response experiments included starting tissue (tissue before culture), a control condition for normalization of data between blots, a DMSO condition (zero concentration of genistein), and genistein at concentrations ranging from 1.0 to 10 µg/ml (Fig. 1). SP-A content increased significantly after 4 days in culture in the control, vehicle (DMSO), and 1.0 µg/ml genistein conditions compared with the undifferentiated start tissue before culture (Fig. 1). There was a clear decrease in the amount of SP-A present in human fetal lung tissue after treatment with 10 µg/ml of genistein for 4 days compared with the DMSO vehicle-only condition (Fig. 1). Quantitation by densitometry revealed that genistein significantly inhibited the amount of SP-A isolated from cultured human fetal lung explants in a dose-dependent manner, with SP-A content decreasing by one-half at a dose of 10 µg/ml (ANOVA, P < 0.0001) (Fig. 1, bottom; Newman-Keuls test, P < 0.05 for genistein >=  5.0 µg/ml, n = 4 experiments).


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Fig. 1.   Effect of genistein (1.0, 5.0, and 10.0 µg/ml; 4-day exposure) on surfactant protein (SP) A content (35-kDa band) in human fetal lung explants. Top: representative SP-A immunoblot showing a clear decrease in SP-A content at 10 µg/ml of genistein compared with DMSO vehicle-only condition. SP-A content increased after 4 days in culture in both control (C) and vehicle [DMSO (D); 0 µg/ml genistein] conditions compared with undifferentiated start tissue (ST) before culture. Bottom: densitometric data of SP-A levels normalized to control, with control value equal to 1 for each experiment. Values are means ± SE; n = 4 experiments. There is a significant dose-dependent decrease in SP-A with increasing concentrations of genistein (P < 0.0001, 1-way ANOVA). * Significant decrease in SP-A at doses of genistein >=  5.0 µg/ml vs. DMSO (Newman-Keuls test, P < 0.05). dagger  Significant increase in SP-A levels between starting tissue and cultured tissue from either control, DMSO, or 1.0 µg/ml of genistein condition (Newman-Keuls test, P < 0.05).

Effect of genistein on steady-state levels of SP-A mRNA. To determine whether the inhibitory effects of genistein occurred at the pretranslational stage, we evaluated SP-A gene expression. We measured steady-state levels of SP-A mRNA isolated from fetal lung explants that had been cultured for 4 days with genistein. The experiments involved a control condition for normalization of data between blots, a DMSO condition (zero concentration of genistein), and genistein (1.0, 5.0, and 10 µg/ml). Analysis of total RNA isolated from the human fetal lung explants by Northern blot identified a 2.1-kb SP-A mRNA, consistent with previously reported human SP-A mRNA transcripts (36). Steady-state levels of SP-A mRNA increased significantly after 4 days in culture in both the control and vehicle (DMSO) conditions compared with the undifferentiated start tissue before culture (Fig. 2, top). In the cultured tissue exposed to genistein, there is a clear decrease in steady-state levels of SP-A mRNA (Fig. 2, top). Densitometric analysis of SP-A mRNA was performed, and the density of each band was normalized to the corresponding 18S rRNA band to control for loading artifact. The densities of the SP-A mRNA bands from each blot were normalized to the control condition, which was set at a value of one, and compared between blots. We found a significant decrease in steady-state levels of SP-A mRNA with exposure to genistein (1-way ANOVA, P < 0.001, n = 3 experiments; Tukey's test, P < 0.05 for genistein >=  5.0 µg/ml; Fig. 2, bottom).


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Fig. 2.   Effect of genistein (0-10.0 µg/ml; 4-day treatment) on steady-state levels of SP-A mRNA isolated from human fetal lung explants. Top: representative autoradiogram of an SP-A Northern blot hybridized with 32P-labeled human SP-A cDNA (10 µg of total RNA/lane). There is a clear decrease in steady-state level of SP-A mRNA at 5 and 10 µg/ml of genistein. Bottom: densitometric analysis of SP-A mRNA was performed, and density of each band was normalized to corresponding 18S rRNA band from photograph of corresponding ethidium-stained RNA gel to control (Ctrl) for loading artifact. Densities of SP-A mRNA bands from each blot were normalized to control condition, which was set at a value of 1, and compared between experiments. Values are means ± SE. Start, starting tissue. There is a significant decrease in steady-state levels of SP-A mRNA with increasing concentrations of genistein (1-way ANOVA, P < 0.05). * Significant decrease in SP-A mRNA at doses of genistein >=  5.0 µg/ml vs. control and at 10.0 µg/ml of genistein vs. either control or DMSO condition (Tukey's test, P < 0.05). dagger  Significant increase in SP-A mRNA between starting tissue and control cultured tissue (Tukey's test, P < 0.05).

Effect of genistein and EGF on SP-A content. We measured the interaction between genistein (10 µg/ml) and EGF (10 ng/ml) on SP-A content in human fetal lung explants cultured for 4 days to see whether genistein could block the known stimulatory effects of EGF on human SP-A synthesis (36). As expected, SP-A content increased significantly in both the control and vehicle conditions compared with the undifferentiated start tissue before culture (Fig. 3). Genistein significantly decreased SP-A content compared with the vehicle (DMSO) condition alone (ANOVA, P < 0.0001; Newman-Keuls test, P < 0.05; Fig. 3). EGF treatment caused a small but significant increase in SP-A compared with the vehicle condition. There was no difference in SP-A content in explants exposed to genistein alone compared with explants exposed to both genistein and EGF, with both conditions having significantly less SP-A than the vehicle (n = 3-4 experiments). Thus the addition of EGF failed to overcome the inhibitory effect of genistein on SP-A synthesis. Daidzein, an inactive analog of genistein, did not significantly reduce SP-A compared with the vehicle.


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Fig. 3.   Effect of genistein with and without epidermal growth factor [EGF (E)] on SP-A (35-kDa band) in human fetal lung explants during 4 days of incubation. Top: representative SP-A immunoblot showing no difference in SP-A content between fetal lung tissue cultured with genistein (G; 10 µg/ml) alone and that cultured with genistein+EGF (10.0 ng/ml). Daidzein (Dz) did not decrease SP-A content compared with vehicle (DMSO). Bottom: densitometric data (means ± SE) of SP-A levels normalized to control, with control value equal to 1 for each experiment. Gen, genistein. There were significant differences among some conditions (ANOVA, P < 0.0001, n = 3-4 experiments). SP-A content significantly increased after 4 days in culture in both control and vehicle (DMSO) conditions compared with undifferentiated start tissue before culture (Newman-Keuls test, dagger  P < 0.05). EGF slightly increased SP-A content compared with vehicle (Newman-Keuls test, # P < 0.05), and both genistein and genistein+EGF significantly decreased SP-A content compared with vehicle (Newman-Keuls test, * P < 0.05).

Toxicity and morphology. Tissue viability was not affected by the doses of genistein used in this study. There was no evidence of cytotoxicity, as measured by the release of LDH into the media from explants cultured for 4 days, with the highest concentration of genistein (10 µg/ml) compared with the vehicle (1.75 ± 0.16 vs. 1.83 ± 0.30 IU/l, means ± SE, n = 4 experiments).

To assess morphological maturation, we examined explants that had been cultured with genistein (10 µg/ml) for 4 days, which was the same length of time in which genistein inhibited SP-A synthesis. Electron microscopy revealed normal developmental changes as seen by the presence of lamellar bodies in type II epithelial cells, with microvilli covering their apical surface (Fig. 4). Similar maturational changes were also observed in electron micrographs of the control and vehicle (DMSO) explants (data not shown). Furthermore, even after 7 days of exposure to high-dose genistein, a time period in which lamellar bodies become abundant in cultured human fetal lung (34), we saw no harmful effects on morphological maturation. Thus genistein did not prevent distal pulmonary epithelial cells from differentiating into cuboidal lamellar body-containing alveolar type II cells, similar to the control and vehicle conditions (Fig. 5).


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Fig. 4.   Electron micrograph of human fetal lung tissue cultured for 4 days with genistein (10 µg/ml) showing a differentiated type II pneumocyte containing lamellar bodies (LB) with microvilli (mv) lining apical surface and extending into ductal lumen (L). Bar = 1 µm.


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Fig. 5.   Electron micrographs of human fetal lung tissue cultured for 7 days. All panels are at same magnification; bar = 3 µm. Top: control condition showing presence of cuboidal type II pneumocytes with abundant lamellar bodies (arrows) lining lumen of a duct. Middle: vehicle (DMSO) condition showing presence of cuboidal type II pneumocytes with abundant lamellar bodies lining lumen of a duct. Bottom: fetal lung tissue exposed to genistein (10 µg/ml) for 7 days also showing presence of cuboidal type II pneumocytes with abundant lamellar bodies lining lumen of a duct. Genistein did not prevent morphological differentiation of distal pulmonary epithelium into alveolar type II cells.

The time course for the inhibitory effect of genistein (10 µg/ml) on SP-A was determined by harvesting explants after 1-4 days in culture. SP-A content was measured by immunoblot, densitometric analysis was performed, and the data were normalized to the day 1 control within each blot. SP-A content increased by more than sevenfold with 4 days in culture in both the control (ANOVA, P = 0.01) and DMSO (vehicle; ANOVA, P = 0.008) conditions (Fig. 6). There was a significant decrease in SP-A content after 3 days of exposure to genistein compared with DMSO, and by 4 days, SP-A content was significantly lower in the genistein group compared with either the control or DMSO condition (Fig. 6).


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Fig. 6.   Time course of effect of genistein on levels of SP-A in human fetal lung explants. Explants were cultured for 1-4 days with either DMSO (vehicle) or genistein (10 µg/ml). SP-A content was measured by densitometry and then normalized within each blot so that day 1 control value was equal to 1. Values are means ± SE; n = 2 experiments. There was a significant increase in SP-A content over 4 days in culture in both control (dagger  P = 0.01, ANOVA) and DMSO (ddager  P = 0.008, ANOVA) conditions. After 3 days of exposure to genistein, SP-A content was significantly decreased compared with DMSO (# P < 0.05, ANOVA, Newman-Keuls test). After 4 days of exposure to genistein, SP-A content was now significantly decreased compared with both control and DMSO condition (* P < 0.05, ANOVA, Newman-Keuls test).

Effect of genistein on basal EGF-receptor tyrosine phosphorylation. To determine whether EGF-receptor tyrosine kinase activity could be decreased by chronic exposure to genistein, we measured basal EGF-receptor tyrosine phosphorylation in human fetal lung explants cultured for 4 days in the presence of genistein at the same concentrations (1-10 µg/ml) that inhibited SP-A. Fresh genistein was added daily to the cultures. Antiphosphotyrosine antibodies were used to identify the presumptive tyrosine-phosphorylated EGF receptor (170 kDa). Immunoblots were quantitated by densitometry. The densitometric data obtained from each experiment were normalized to the control condition, with the control value equal to one. Notably, at the doses of genistein used for our experiments, there was no effect on basal EGF-receptor tyrosine phosphorylation (Fig. 7). The start tissue before culture equaled 1.04 ± 0.08, control equaled 1.0, vehicle (DMSO) equaled 0.84 ± 0.12, genistein at 10 µg/ml equaled 0.81 ± 0.06, genistein at 5 µg/ml equaled 0.84 ± 0.05, and genistein at 1 µg/ml equaled 0.88 ± 0.02 (means ± SE, n = 3 experiments, no significant effect by 1-way ANOVA).


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Fig. 7.   Effect of chronic genistein treatement on endogenous EGF-receptor tyrosine phosphorylation. Representative immunoblot of tyrosine-phosphorylated EGF receptor (170-kDa band) with the use of antiphosphotyrosine antibodies to detect EGF-receptor phosphotyrosine residues in human fetal lung tissue cultured for 4 days with genistein added daily. Genistein did not decrease basal levels of tyrosine-phosphorylated EGF receptor in cultured fetal lung explants. Lane 1, molecular-mass markers; lane 2, starting tissue before culture; lane 3, control; lane 4, DMSO vehicle; lane 5, genistein (10 µg/ml); lane 6, genistein (5 µg/ml); lane 7, genistein (1 µg/ml). For densitometric data see RESULTS.

Effect of tyrphostin AG-1478 on basal EGF-receptor tyrosine phosphorylation. To determine whether EGF-receptor tyrosine kinase activity could be decreased by chronic exposure to a more specific EGF-receptor tyrosine kinase inhibitor, we measured basal levels of tyrosine-phosphorylated EGF receptor in human fetal lung explants cultured for 4 days with tyrphostin AG-1478 (10 µg/ml, 30 µM) (Fig. 8). The phosphotyrosine immunoblots (n = 3) were quantitated by densitometry and normalized to the control condition, which was made equal to one. The start tissue before culture equaled 1.76 ± 0.18, control equaled 1.0, vehicle (DMSO) equaled 0.93 ± 0.01, EGF (10 ng/ml) equaled 1.05 ± 0.23, tyrphostin (30 µM) equaled 0.26 ± 0.12, tyrphostin (30 µM) plus EGF (10 ng/ml) equaled 0.04 ± 0.08, and tyrphostin A1 (30 µM, inactive analog) equaled 1.35 ± 0.33 (means ± SE). There was a significant reduction in presumptive phosphorylated EGF-receptor tyrosine (170,000-Da band; Fig. 8, lane 6) in human fetal lung explants exposed to tyrphostin (1-way ANOVA, P < 0.0001) compared with the vehicle or control conditions (Newman-Keuls test, P < 0.05). The addition of EGF did not overcome the inhibitory effect of tyrphostin on tyrosine-phosphorylated EGF receptor (lane 7 vs. lane 6). The inactive analog (tyrphostin A1, 30 µM, lane 8) did not significantly affect the level of tyrosine-phosphorylated EGF receptor compared with either the vehicle or control condition. The starting tissue had significantly more phosphorylated EGF receptor than either the control or vehicle tissue that had been cultured for 4 days (Newman-Keuls test, P < 0.05).


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Fig. 8.   Effect of chronic tyrphostin AG-1478 treatment on endogenous EGF-receptor tyrosine phosphorylation. Top: representative immunoblot of tyrosine-phosphorylated EGF receptor (EGF-R; 170-kDa band) using antiphosphotyrosine antibodies to detect EGF-receptor phosphotyrosine residues in human fetal lung tissue cultured for 4 days with tyrphostin (T; 30 µM) added daily. Tyrphostin clearly decreased basal level of presumptive EGF-receptor tyrosine phosphorylation, and addition of EGF did not overcome inhibitory effect of tyrphostin on tyrosine-phosphorylated EGF receptor in cultured fetal lung explants. Tyrphostin A1 (A1), an inactive analog, did not significantly decrease tyrosine-phosphorylated EGF receptor. Lane 1, molecular-mass markers; lane 2, starting tissue before culture; lane 3, control; lane 4, DMSO vehicle; lane 5, EGF (10 ng/ml); lane 6, tyrphostin AG-1478 (30 µM); lane 7, tyrphostin+EGF; lane 8, tyrphostin A1 (30 µM; inactive analog); lane 9, tyrosine-phosphorylated EGF-receptor standard from A-431 cells (positive control). Bottom: densitometric data (means ± SE; n = 3 experiments) of tyrosine-phosphorylated EGF receptor normalized to control, with control value equal to 1 for each experiment. There were significant differences among conditions (P < 0.0001, ANOVA). Tyrphostin (Tyrph) and tyrphostin+EGF significantly decreased level of phosphorylated EGF receptor compared with vehicle (DMSO), control, and tyrphostin A1 (inactive analog) conditions (*# P < 0.05, Newman-Keuls test). There was no significant difference between either control or vehicle condition and tyrphostin A1. Starting tissue had significantly more phosphorylated EGF receptor than control or vehicle tissue that had been cultured for 4 days (Newman-Keuls test, dagger  P < 0.05).

Effect of tyrphostin AG-1478 on SP-A content. Immunoreactive SP-A was measured in human fetal lung explants that were cultured for 4 days in the same concentration of tyrphostin AG-1478 (30 µM) that had been previously shown to inhibit phosphorylation of EGF-receptor tyrosine. The SP-A immunoblots (n = 4-5) were quantitated by densitometry and normalized so that the control condition equaled one. The start tissue before culture equaled 0.14 ± 0.07, vehicle (DMSO) equaled 1.03 ± 0.06, EGF (10 ng/ml) equaled 1.13 ± 0.22, tyrphostin (30 µM) equaled 0.13 ± 0.03, tyrphostin (30 µM) plus EGF (10 ng/ml) equaled 0.19 ± 0.04, and tyrphostin A1, an inactive analog, equaled 0.55 ± 0.10 (means ± SE). Tyrphostin AG-1478 significantly decreased SP-A content (1-way ANOVA, P < 0.0001; Fig. 9) compared with the vehicle (Newman-Keuls test, P < 0.05). Furthermore, the addition of EGF did not overcome the inhibitory effect of tyrphostin on the level of SP-A (lane 7 vs. lane 6).


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Fig. 9.   Effect of tyrphostin AG-1478 on SP-A (35-kDa band). Top: representative immunoblot of SP-A isolated from human fetal lung explants cultured for 4 days with tyrphostin AG-1478. Lane 1, molecular-mass markers; lane 2, starting tissue before culture; lane 3, control; lane 4, DMSO (vehicle); lane 5, EGF (10 ng/ml); lane 6, tyrphostin (30 µM); lane 7, tyrphostin (30 µM) plus EGF (10 ng/ml); lane 8, tyrphostin A1 (30 µM; negative tyrphostin control); lane 9, SP-A standard. SP-A was clearly decreased by tyrphostin AG-1478, and addition of exogenous EGF did not overcome tyrphostin-induced inhibition of SP-A content. Bottom: densitometric data (means ± SE) of SP-A levels normalized to control, with control value equal to 1 for each experiment. There were significant differences among conditions (ANOVA, P < 0.0001, n = 4-5 experiments). SP-A content increased significantly after 4 days in culture in control, vehicle (DMSO), and EGF conditions compared with undifferentiated start tissue before culture (Newman-Keuls test, dagger  P < 0.05). Both tyrphostin and tyrphostin+EGF significantly decreased SP-A content compared with vehicle (Newman-Keuls test, * P < 0.05) and compared with inactive analog tyrphostin A1 (Newman-Keuls test, # P < 0.05).

The release of LDH into the media from explants cultured for 4 days with 30 µM tyrphostin AG-1478 was measured to assess toxicity and compared with control after being normalized to the vehicle. There was no significant difference in LDH levels between the control condition and tyrphostin (1.05 ± 0.12 vs. 1.21 ± 0.05 IU/l, means ± SD, n = 3 experiments).

Effect of tyrphostin AG-1478 on steady-state levels of SP-A mRNA. To determine whether the inhibitory effects of tyrphostin occurred at the pretranslational stage, we measured steady-state levels of SP-A mRNA isolated from fetal lung explants that had been cultured for 4 days in the same concentration of tyrphostin AG-1478 (30 µM) that had been previously shown to decrease SP-A content. Steady-state levels of SP-A mRNA increased significantly after 4 days in culture in both the control and vehicle (DMSO) conditions compared with the undifferentiated start tissue before culture (Fig. 10). In the cultured tissue exposed to tyrphostin, there was a clear decrease in steady-state levels of SP-A mRNA, and the addition of EGF did not overcome the inhibitory effect of tyrphostin (Fig. 10). Densitometric analysis of SP-A mRNA was performed, and the data were normalized within each lane to 18S rRNA to control for loading. SP-A mRNA levels were normalized to the control condition, with the control condition set equal to one and compared between blots. We found a significant decrease in steady-state levels of SP-A mRNA with exposure to tyrphostin (1-way ANOVA, P < 0.0001, n = 5 experiments; Fig. 10, bottom). Tyrphostin A1, an inactive analog, did not decrease SP-A mRNA compared with the vehicle (DMSO).


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Fig. 10.   Effect of tyrphostin AG-1478 (30 µM; 4-day treatment) on steady-state levels of SP-A mRNA isolated from human fetal lung explants. Top: representative autoradiogram of Northern blot of total RNA (10 µg/lane) hybridized with 32P-labeled human SP-A cDNA, stripped, and reprobed with 32P-labeled human 18S ribosomal cDNA. Lane 1, starting tissue before culture; lane 2, control; lane 3, DMSO (vehicle); lane 4, EGF (10 ng/ml); lane 5, tyrphostin (30 µM); lane 6, tyrphostin (30 µM) plus EGF (10 ng/ml); lane 7, tyrphostin A1 (30 µM; negative tyrphostin control). There is a clear decrease in level of SP-A mRNA in explants treated with tyrphostin that is not affected by addition of EGF. Bottom: densitometric data (means ± SE) of steady-state levels of SP-A mRNA normalized to 18S rRNA in each lane to control for loading artifact and then normalized to control condition for each experiment, with control value equal to 1. There is a significant decrease in steady-state levels of SP-A mRNA with tyrphostin, regardless of presence of EGF compared with either control or tyrphostin A1 (inactive analog) (1-way ANOVA, Newman-Keuls test, *# P < 0.05). dagger  Significant increase in SP-A mRNA between starting tissue and control cultured tissue (Newman-Keuls test, P < 0.05).

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Modulation of SP-A levels is important for normal human lung function. SP-A has many roles, including helping to form tubular myelin, aiding the surface tension-reducing properties of surfactant phospholipids, regulating the recycling and secretion of surfactant phospholipids, and contributing to the immune system defense of the lung (35). EGF has previously been shown to increase human SP-A synthesis and expression in human fetal lung explants (36). EGF mediates its effects through a cell-surface receptor that is dependent on intrinsic tyrosine kinase activity to engage its signal transduction cascade, making intact tyrosine kinase activity critical for EGF-receptor function (6). Thus we hypothesized that EGF-receptor tyrosine kinase activity is involved in the regulation of SP-A synthesis during human fetal lung development.

To study the effects of tyrosine kinase inhibition on the developmental regulation of human SP-A, we blocked tyrosine kinase activity in human tissue using cultured fetal lung explants as an in vitro model of fetal lung development. It has previously been shown that midtrimester undifferentiated human fetal lung tissue will spontaneously differentiate into alveolar type II cells with the capacity to produce SP-A after 3-4 days in explant culture (27, 29). Previously, we detected EGF receptor in the cell membranes of human alveolar epithelium from spontaneously differentiating cultured fetal lung explants (17). The presence of EGF receptor in distal lung epithelium supports a role for EGF-receptor tyrosine kinase in the development and regulation of SP-A synthesis. Thus we tested the hypothesis that inhibition of EGF-receptor function by blocking tyrosine kinase activity would decrease the expression of human SP-A. We evaluated the effect of genistein [a broad-range inhibitor of tyrosine kinases (1, 2, 21)] and tyrphostin AG-1478 [a highly specific inhibitor of EGF-receptor tyrosine kinase (20)] on SP-A content and SP-A mRNA in spontaneously differentiating human fetal lung explants cultured for 4 days in the presence of these agents.

We found that genistein significantly decreased SP-A content in differentiating fetal lung explants in a dose-dependent manner. The significant increase in SP-A content after 4 days of culture in the control and vehicle conditions compared with the undifferentiated start tissue validated the use of this model (27) to study the effects of tyrosine kinase inhibitors during human type II cell differentiation. Genistein also inhibited SP-A mRNA expression, implying an effect at the pretranslational level. Furthermore, the inhibitory effect of genistein on SP-A content could not be overcome by the addition of exogenous EGF, implying a block in the EGF-receptor signal transduction pathway.

The inhibitory effect of genistein on SP-A content in fetal lung explants was not a result of toxicity, since exposure to genistein did not increase the release of LDH into the media. Morphological maturation also remained unaffected, since chronic exposure of the fetal lung explants to genistein for 4-7 days did not interfere with the normal differentiation of the distal pulmonary epithelium into type II pneumonocytes.

To determine whether the doses of genistein used to inhibit SP-A synthesis acted through the EGF-receptor tyrosine kinase, we measured basal levels of tyrosine-phosphorylated EGF receptor in explants cultured in the presence or absence of genistein. To our surprise, at the maximum dose of genistein used, there was no effect on basal presumptive EGF-receptor tyrosine phosphorylation, suggesting an effect on tyrosine kinase other than the EGF receptor in modulating SP-A synthesis. Thus to more specifically evaluate the role of the EGF receptor in the regulation of SP-A synthesis, we needed to measure SP-A levels after exposure to an inhibitor that could block EGF-receptor tyrosine phosphorylation in human fetal lung explants. Thus we treated the explants with a second inhibitor, tyrphostin AG-1478, which is highly specific to EGF-receptor tyrosine kinase (20).

We found that exposure to tyrphostin AG-1478 significantly reduced both basal and EGF-induced levels of tyrosine-phosphorylated EGF receptor in human fetal lung explants. Thus, using tyrphostin, we evaluated the effect of inhibiting EGF-receptor tyrosine kinase on SP-A metabolism during human fetal lung development in vitro. We found that tyrphostin significantly decreased both SP-A content and SP-A mRNA at the same concentration that inhibited basal tyrosine phosphorylation of EGF receptor. The addition of EGF failed to overcome the inhibitory effects of tyrphostin on SP-A metabolism. Our finding that inhibition of EGF-receptor activity leads to decreased levels of human SP-A agrees with the detection of decreased SP-A immunostaining in the lungs of EGF-deficient neonatal rats (22).

Although the two inhibitors used in the above experiments have different mechanisms of action [genistein acts at the binding site for ATP for tyrosine kinases (1, 2, 21) and tyrphostin acts at the substrate-binding site for EGF-receptor tyrosine kinase (20)], both significantly decreased SP-A content in human fetal lung explants. In developing human fetal lung tissue, there must exist distinct tyrosine kinase-dependent signal transduction pathways for the regulation of SP-A synthesis, including but not limited to a pathway initiated through the EGF receptor. Furthermore, because global inhibition of protein tyrosine kinase activity with genistein also blocks SP-A expression without directly inhibiting EGF-receptor tyrosine phosphorylation, there must exist additional protein tyrosine kinases downstream from the EGF receptor that play an important role in regulating SP-A synthesis. Potential candidates for this role include nonreceptor protein tyrosine kinases from the Src or Jak family that are known to be involved in the signaling pathways of numerous cytokines (32). For example, the 50% inhibitory dose for genistein to inhibit pp60v-src tyrosine phosphorylation is 7.0 µg/ml (2), which is within the dose range that inhibited SP-A expression in our experiments.

The inhibitory effect of genistein on SP-A expression could also have resulted from the inhibition of receptor protein tyrosine kinases other than the EGF receptor. These include the insulin receptor, the platelet-derived growth factor (PDGF) receptor, the insulin-like growth factor I (IGF-I) receptor, the fibroblast growth factor (FGF) receptor, the colony-stimulating factor-1 (CSF-1) receptor, and the nerve growth factor receptor (3). For example, genistein at a dose of 100 µM inhibits both PDGF-induced DNA synthesis and PDGF-induced phospholipase C activation in mouse fibroblasts (2). Ligands for some of these receptors [e.g., PDGF (15), IGF-I (26), and FGF (14)] have been found in lung tissue, implying the presence of their receptors. Thus it is possible that non-EGF-receptor protein tyrosine kinases may have been affected by genistein; however, the low doses of genistein used make this less likely compared with the known lower dose effects of genistein on nonreceptor protein tyrosine kinases.

Serine and threonine kinases can also be inhibited by genistein (2). This effect was unlikely in our experiments, since the concentration of genistein needed for 50% inhibition of known serine or threonine protein kinases (e.g., cAMP-dependent protein kinase, protein kinase C, phosphorylase kinase, 5'-nucleotidase, and phosphodiesterase) is >370 µM (2), which is 10-fold greater than the maximal concentration of genistein used here. Genistein has effects on other areas of cellular metabolism that do not involve tyrosine phosphorylation such as mitochondrial pyruvate transport and respiratory chain activity (38). However, the concentration needed to achieve at least 60% inhibition of pyruvate oxidation, 200 µM (38), is still fivefold greater than the maximal dose of genistein (37 µM) used. Thus this suggests that the effects of genistein on SP-A were primarily due to inhibition of tyrosine kinase activity other than EGF-receptor tyrosine kinase.

There was a time-dependent effect of genistein on SP-A content in cultured human fetal lung explants in which 3-4 days passed before a significant decrease in SP-A levels occurred. We believe that this time frame is consistent with decreased transcription of SP-A both during and after the occurrence of alveolar type II cell differentiation. We speculate that genistein blocks a tyrosine kinase-dependent signal transduction cascade, which results in a decreased expression of regulatory transcription factors such as c-Fos and c-Jun (33). However, this speculation will need further study.

The use of a human fetal lung explant model was necessary to study the effects of tyrosine kinase inhibition on SP-A metabolism during the critical period of alveolar type II cell differentiation. However, this model does not allow for clear delineation of cell-specific effects. We have shown using the explant model that the EGF receptor is located in alveolar epithelium during type II cell differentiation (17). Furthermore, we have shown that genistein inhibition of tyrosine kinase activity directly inhibits expression of SP-A in pulmonary epithelial cells (18). These experiments were conducted using a cell-specific model, the H441 cell, which is a human pulmonary adenocarcinoma cell line in which the cells synthesize and secrete both SP-A and SP-B and have morphology characteristic of pulmonary epithelial cells (19). Still, this does not rule out the possibility of genistein inhibiting tyrosine kinases within mesenchymal as well as epithelial cells within the explants, with the resultant decrease in SP-A from changes in cell-to-cell communication rather than a direct effect on the epithelial cells alone.

We conclude that specific inhibition of EGF-receptor tyrosine kinase with tyrphostin AG-1478 decreases SP-A expression during human fetal lung development and that general tyrosine kinase inhibition with genistein also decreases SP-A expression and blocks the effects of EGF without inhibiting EGF-receptor tyrosine phosphorylation. The doses of genistein used should have minimized its effects on other receptor protein tyrosine kinases, but nonreceptor protein tyrosine kinases may have been affected. The present study suggests that a tyrosine kinase-dependent signal transduction pathway, including but not limited to the EGF receptor at the point of initiation, is critical to the regulation of SP-A during human fetal lung development.

    ACKNOWLEDGEMENTS

This work was supported by Clinical Research Grant 6-FY95-0992 from the March of Dimes Birth Defects Foundation, a research grant from the American Lung Association, and National Heart, Lung, and Blood Institute Grant R29-HL-52055.

    FOOTNOTES

Address for reprint requests: J. M. Klein, Dept. of Pediatrics, Univ. of Iowa, 200 Hawkins Dr., Iowa City, Iowa 52242-1083.

Received 10 April 1997; accepted in final form 14 January 1998.

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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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