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Endocrinology Vol. 141, No. 8 2923-2929
Copyright © 2000 by The Endocrine Society

ARTICLES

Androgen Regulation of Signaling Pathways in Late Fetal Mouse Lung Development1

Christiane E. L. Dammann, Sujatha M. Ramadurai, Dana D. McCants, Lucia D. Pham and Heber C. Nielsen

Department of Pediatrics, Division of Newborn Medicine, Tufts University (C.E.L.D., S.M.R., D.D.M., L.D.P., H.C.N.), Boston, Massachusetts 02111; and Department of Cell Biology, Harvard Medical School, Division of Signal Transduction, Beth Israel Deaconess Medical Center (C.E.L.D.), Boston, Massachusetts 02215

Address all correspondence and requests for reprints to: Christiane E. L. Dammann, M.D., Department of Pediatrics, Division of Newborn Medicine, Tufts University, Boston, Massachusetts 02111. E-mail: cdammann{at}lifespan.org


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
During lung development there is tension between positive and negative regulators of fibroblast-epithelial communication controlling type II cell differentiation. A clinical consequence of imbalance of this tension is the increased risk for respiratory distress syndrome in male infants. We hypothesized that chronic intrauterine androgen exposure alters fetal lung fibroblast maturation by down-regulating epidermal growth factor receptor (EGF-R) activity and by up-regulating transforming growth factor-ß receptor (TGFß-R) activity, leading to an inhibition of surfactant protein B (SP-B) and -C (SP-C) gene expression in type II cells. We treated pregnant mice with dihydrotestosterone (DHT; 2 mg/day) or vehicle for 7 days, starting on gestational day 11. On day 18, EGF binding, EGF-R phosphorylation, TGFß-R binding, and TGFß1-induced cell proliferation were studied in sex-specific fibroblast cultures. SP-B and -C messenger RNA levels were measured in whole lungs. Chronic DHT treatment reduced both EGF binding (females to 78 ± 8% and males to 65 ± 9% of controls) and EGF-induced EGF-R phosphorylation. TGFß-R binding was increased (females to 173 ± 39% and males to 280 ± 64% of controls), and TGFß-induced cell proliferation was increased in female cells (231 ± 57% of controls). SP-B and -C messenger RNA expression was reduced to 55 ± 10% and 75 ± 4%, respectively. We conclude that chronic DHT exposure beginning early in lung development alters the balance of growth factor signaling that regulates lung maturation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE DELAY IN surfactant synthesis (1) as well as the increased risk for the development of respiratory distress syndrome (RDS) (2, 3) in the male fetus are well known. Androgens have been implicated in the mechanism of delay in lung maturation. Treatment with dihydrotestosterone (DHT) during sexual differentiation inhibits surfactant phospholipid production in the female fetus, whereas treatment with the antiandrogen flutamide increases surfactant phospholipid production in the male fetus (4). Several studies show that androgens act on the fibroblast to delay the development of fibroblast-type II cell communication (5, 6, 7). The delay in the male fetus and the response of the fetal lung to androgen are dependent on the presence of functional androgen receptors in the fetal lung (6, 8).

The onset of surfactant synthesis is regulated by fibroblast-type II cell communication (9, 10, 11, 12, 13, 14, 15). The fetal lung fibroblast produces a differentiation factor, which stimulates the type II cell to mature and produce surfactant (16). This process of lung cell maturation is known to be influenced by many hormones and growth factors (9).

Epidermal growth factor (EGF) is a potent mitogen that stimulates cell proliferation and cell differentiation (17, 18, 19). EGF promotes fetal lung type II cell maturation by advancing fibroblast-type II cell communication through specific stages of development (20). EGF causes stimulation of type II cell disaturated phosphatidylcholine (DSPC) synthesis and surfactant protein A and C (SP-A, SP-C) expression (12, 21). EGF receptor (EGF-R) activity peaks in fetal lung fibroblasts at the gestation corresponding to the onset of fibroblast-type II cell communication that stimulates fetal lung surfactant synthesis. This peak in EGF-R activity occurs later in gestation in the male fetus (22).

Transforming growth factor-ß1 (TGFß1), a cellular mitogen, is produced by immature fetal lung fibroblasts (23). TGFß1 inhibits fetal lung maturation by inhibiting fibroblast-type II cell communication, type II cell DSPC synthesis, and SP-A and SP-C expression (12, 23, 24, 25). TGFß1 exerts development-specific effects on lung fibroblasts, stimulating adult, but inhibiting immature, fetal lung fibroblast proliferation (26). It has been speculated that androgens mediate their inhibitory effect on lung maturation through TGFß1 mechanisms (23). For example, androgen inhibition of fibroblast-type II cell communication has been reversed with antibodies to TGFß1 (23). Because the fetal lung expresses TGFß receptor (TGFß-R), the inhibitory effect of TGFß1 on lung maturation must be overcome with advancing gestation. We previously reported that TGFß-R binding and total receptor number decrease in late gestation fetal lung fibroblasts. This decrease happened earlier in gestation in female cells. Furthermore, late gestation female fibroblasts treated with androgens showed increased TGFß-R binding. All of these events were accompanied by an alteration in the relative proportions of receptor subtypes and an altered proliferative response to TGFß1 stimulation (27).

The androgen regulation of signaling pathways in the process of fetal lung maturation, especially the effect on the regulation of EGF-R and TGFß-R, is unknown. We proposed that the inhibitory effect of androgens on the development of fibroblast-type II cell communication is mediated by an alteration of the developmental changes in these receptors. Specifically, we hypothesized that chronic intrauterine androgen exposure down-regulates EGF binding and EGF-R phosphorylation and up-regulates TGFß-R binding and TGFß1-induced cell proliferation in fetal lung fibroblasts, with consequent inhibition of type II cell differentiation, reflected by inhibition of SP-B and -C messenger RNA (mRNA) expression. We therefore studied the effect of androgen on these signaling pathways in the fetal lung. Exogenous DHT treatment in a supraphysiological dose (4) started early in lung development was used to delay the maturational process in both sexes. The effect of DHT on EGF-R and TGFß-R activity was measured in late gestation when differences in maturation have resolved (28, 29), and the baseline activities of these receptors are similar in both sexes (22, 27).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
Time-dated pregnant Swiss-Webster mice (Taconic Farms, Inc., Germantown, NY) were used, in whom the day of mating was denoted day 0 of gestation. The following reagents were purchased as indicated: culture medium, HBSS, trypsin, TRIzol reagent, deoxyribonuclease, culture dishes, and antihuman EGF-R (sheep polyclonal antibody) from Life Technologies, Inc.(Grand Island, NY); six-well tissue culture plates from Falcon/Becton Dickinson and Co. (Franklin Lakes, NJ); FCS from HyClone Laboratories, Inc. (Logan, UT); porcine TGFß1 from R&D Systems (Minneapolis, MN); DHT from Steraloids (Wilton, NH); [125I]EGF (SA, 92–180 mCi/mg) and [3H]thymidine (SA, 50 Ci/mmol) from ICN Biochemicals, Inc. (Irvine, CA); [125I]TGFß1 (SA, 93–173 mCi/mg) from NEN Life Science Products (Cambridge, MA); 32P-labeled nucleotides (SA, 3000 µCi/µg) from ICN Biochemicals, Inc.; BSA from Calbiochem (La Jolla, CA); HEPES, Triton X-100, aprotinin, leupeptin, pepstatin, phenylmethylsulfonylfluoride (PMSF), mouse EGF (receptor grade), and mol wt standards from Sigma (St. Louis, MO); protein A-Sepharose CL-4B from Amersham Pharmacia Biotech (Piscataway, NJ); antisheep IgG antibody linked to horseradish peroxidase from Zymed Laboratories, Inc. (South San Francisco, CA); GeneScreen Plus and Renaissance Enhanced Luminol kit from NEN Life Science Products (Boston, MA); Alzet minipumps from Alza Corp. (Palo Alto, CA); DHT time release pellets from Innovative Research of America (Sarasota, FL); Protogel from National Diagnostics (Atlanta, GA); and recombinant horseradish peroxidase-linked antiphosphotyrosine antibody RC 20 from Transduction Laboratories, Inc. (Lexington, KY).

Fetal mouse lung fibroblast cultures
The animal research protocol was approved by the institutional animal research committee. Time-dated pregnant mice were treated for 7 days with a continuous infusion of 2 mg/day DHT or with vehicle using either Alzet minipumps or DHT pellets implanted on day 11 of gestation as described previously (30, 31). This dose of DHT was chosen because a similar dose was used in mice to study androgen effects on fetal sexual differentiation (32) and to document androgen effects on the fetal lung (4, 8). Animals were killed by CO2 inhalation on gestational day 18. The uterus was removed under sterile conditions by laparotomy and kept on ice. Fetuses were kept in DMEM on ice, and fetal sex was identified by the method of Nielsen and Torday (33). The lungs were removed en block under a laminar airflow hood. Some lungs were frozen on liquid nitrogen for RNA extraction. Fibroblast cell cultures were prepared using procedures previously described (20, 22, 27, 34). The lungs were put into sterile HBSS, pooled separately according to sex, and minced into 1-mm3 pieces with a razor blade. The minced lungs were dissociated in HBSS containing deoxyribonculease and 250 mg trypsin in a 37 C water bath for 10–12 min using a stirring bar to disrupt the tissue physically. The reaction was stopped by adding ice-cold DMEM containing 10% charcoal-stripped FCS (FCS-). The cells were filtered through a sterile 70-µm pore size cell strainer and centrifuged at 650 x g for 10 min at 4 C. The pellet was resuspended in DMEM containing 10% FCS- and plated in six-well plates (binding assays), 24-well plates (thymidine incorporation assay), or 100-mm culture dishes (immunoprecipitation) for 60 min at 37 C to allow for differential adherence of lung fibroblasts. The medium was changed to DMEM with 10% FCS-, with DHT (10-8 M) added to cultures from lungs treated with DHT in utero. This dose of 10-8 M was chosen based on dose-response curves performed in earlier studies (34), although the physiological levels of DHT in the amniotic fluid or plasma may be somewhat lower (35, 36). During growth the medium was changed every 24 h, and the cells were kept in culture for 4–5 days for the binding studies and protein determination. Thymidine incorporation was started earlier, after approximately 2–3 days, when the cells reached 50% confluence.

[125I]EGF binding assay
EGF binding was measured using methods previously described by us (22, 34). For each treatment condition, fibroblast cultures at 90–95% confluence were washed twice with 2 ml prewarmed DMEM and incubated in serum-free DMEM for 30 min at 37 C. Nonspecific binding was determined in triplicate using wells containing 1 ml DMEM that were first incubated in a 500-fold excess (200 ng/ml) of unlabeled EGF (receptor grade) for 20 min at room temperature. At the end of this incubation period, 0.4 ng/ml [125I]EGF was added to all wells (nonspecific and total binding). After 60-min incubation at room temperature, the cells were washed three times with 2 ml ice-cold PBS (pH 7.4), to remove the unbound EGF and scraped in 1 ml PBS. Radioactivity was measured using a {gamma}-counter. In each experiment specific EGF binding was measured in two or three wells and was determined by subtracting the radioactivity of the mean nonspecific binding (average binding in triplicate wells preincubated with unlabelled EGF) from the total binding (binding in wells not preincubated with unlabeled EGF). DNA was measured as deoxyribose content in duplicate aliquots (37).

Immunoprecipitation and Western blotting of the EGF-R
Female fibroblast cultures were grown in 100-mm culture dishes in DMEM with 10% FCS- until they reached 80–90% confluence. After a 24-h serum starvation, cells were rinsed and stimulated for 2 min with EGF (100 ng/ml) or DMEM (controls). Cells were then washed with ice-cold PBS and lysed in lysis buffer [20 mM Tris (pH 7.4), 150 mM NaCl, 1 mM MgCl2, 1% Nonidet P-40, 10% glycerol, 1 mM Na3VO4, 1 mM NaF, 1 mM ZnCl2, 10 mM ß-glycerolphosphate, 5 mM tetrasodium pyrophosphate, 1 mM PMSF, and 4 µg/ml each of aprotinin, leupeptin, and pepstatin]. Lysates were cleared by microcentrifugation for 10 min at 4 C, and 200 µg total protein, quantified by the Lowry assay (38), were immunoprecipitated with anti-EGF-R or antiphosphotyrosine antibody (as indicated in the figure legends) for 1.5 h at 4 C with gentle rocking. Protein A-Sepharose was added to each sample, and the incubation was continued for an additional 1.5 h at 4 C. Beads were collected by microcentrifugation and were washed three times in immunoprecipitation buffer [20 mM Tris (pH 7.4), 150 mM NaCl, 1 mM MgCl2, 1% Nonidet P-40, 10% glycerol, 1 mM Na3VO4, 1 mM NaF, 10 mM ß-glycerolphosphate, 5 mM tetrasodium pyrophosphate, 0.2 mM PMSF, and 4 µg/ml each of aprotinin, leupeptin, and pepstatin]. Beads were boiled in Laemmli sample buffer, and proteins were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes. Blots were blocked in 1% BSA in Tris-buffered saline with 0.1% Tween-20 for 1 h at room temperature, incubated with antiphosphotyrosine antibody PC20 overnight at 4 C, and washed three times with Tris-buffered saline/Tween, and proteins were visualized by enhanced chemiluminescence using x-ray film.

[125I]TGFß-R binding assay
The assay was performed as previously described by us with modifications (27). Cultures (at 90–95% confluence) were washed with and then incubated in dissociation buffer (DMEM containing 25 mM HEPES and 0.1% BSA) for 2 h at 37 C to allow for dissociation or internalization of endogenous bound TGFß1. The cells were then washed with and preincubated for 30 min at 4 C in ice-cold binding buffer (128 mM NaCl, 5 mM KCl, 5 mM MgSO4, 1.2 mM CaCl2, 50 mM HEPES, and 10 mg/ml BSA, pH 7.5), after which 50 pM [125I]TGFß1 with or without a 500-fold excess of unlabeled TGFß1 (to determine nonspecific binding) was added. Experiments were performed in triplicate wells. All binding experiments were performed at 4 C to minimize internalization and/or degradation of radiolabeled TGFß1. After an incubation period of 4 h on a rotary shaker (150 rpm), the unbound [125I]TGFß1 was removed by washing with binding buffer. Bound [125I]TGFß1 was solubilized with solubilizing buffer [1% (vol/vol) Triton X-100, 10% (vol/vol) glycerol, 25 mM HEPES, and 10 mg/ml BSA] and counted in a {gamma}-counter. The average nonspecific binding was subtracted from the total binding to yield the specific binding. Additional wells were used to measure the amount of DNA per well on the same plate (37). Specific binding was expressed as counts per min/nmol DNA.

[3H]Thymidine incorporation
Mitogenesis was measured using [3H]thymidine incorporation as described by Zhou to measure the effect of DHT on TGFß-induced cell proliferation (26). At 50% confluence, cells were serum starved for 24 h, treated with 5 µg/ml TGFß1 for 20 h, and pulsed with 2 µCi/ml [3H]thymidine. After cells were rinsed three times with ice-cold PBS, they were disrupted using trypsinization. An aliquot was used to assay the total DNA concentration (37). Incorporated [3H]thymidine was measured by ß-counter. The results were expressed as disintegrations per min/nmol DNA.

Northern blot analysis of surfactant protein mRNA
Rat complementary DNA (cDNA) probes for SP-B and SP-C were previously cloned by us using RT-PCR, as described in detail (39). Total RNA was isolated using TRIzol reagent. Fifteen micrograms of RNA were then size fractionated on a 1% agarose formaldehyde gel and transferred onto GeneScreen Plus by capillary action. After baking the blots at 80 C for 2 h, the Northern blots were prehybridized at 42 C for 2 h in 50% formamide, 5 x SSPE (containing 750 mM NaCl, 150 mM NaH2PO4, and 6 mM EDTA, pH 7.4), 5 x Denhardt’s solution, 1% SDS, 10% dextran sulfate, and 100 µg/ml denatured sheared salmon sperm DNA. The blots were then hybridized in the same solution containing 32P-labeled cDNA probe (prepared by random primer labeling with SA >5 x 108 dpm/µg) at 42 C for 16–20 h. After hybridization the blots were washed twice in 2 x SSPE at room temperature for 15 min each time, followed by a 45-min wash in 2 x SSPE containing 2% SDS at 65 C. Some blots were washed in 0.1 x SSPE containing 0.1% SDS at room temperature for 15 min to reduce nonspecific hybridization. Blots were stripped at 100 C in 0.1% SSC containing 1% SDS for 15 min and then hybridized with 32P-labeled ribosomal protein L32 (RPL32) under the hybridization conditions described above. The blots were analyzed and quantified using a phosphorimager, and the results are expressed as the ratio of surfactant protein mRNA to RPL32 mRNA.

Data analysis
Binding and thymidine incorporation data were expressed as a percentage of their intraexperimental control values. Because our initial hypothesis was specific about the changes expected (decreased EGF binding and SP-B and SP-C expression, increased TGFß binding, and TGFß1-induced thymidine incorporation in DHT-treated cells), a one-tailed t test was used to evaluate the results.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Continuous intrauterine DHT exposure begun early in lung development reduced specific EGF binding in late gestation lung fibroblasts (Fig. 1Go). In female cells, binding was reduced to 79 ± 8% (mean ± SEM) of experiment-specific control values (P < 0.05). Even though male fetuses have higher endogenous DHT levels, there was no difference in the response between females and males. Chronic DHT exposure resulted in a similar reduction of EGF-specific binding in male fibroblasts to 65 ± 9% of that in control cells (P < 0.05). As expected there was no significant difference in specific EGF binding in untreated fibroblasts of both sexes at this gestational age (female cells, 252 ± 43 cpm/nmol DNA; male cells, 365 ± 88 cpm/nmol DNA).



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  		Figure 1. The effect of chronic DHT (gray bars) exposure on specific [125I]EGF binding in fetal day 18 mouse lung fibroblasts compared with experiment-specific controls (black bars). Females, n = 25–26; males, n = 17–18. Values are the mean ± SEM. *, P < 0.05.

 
EGF-R function was studied further by evaluating the induction of EGF-R phosphorylation by EGF. Control and DHT-treated female fibroblasts were immunoprecipitated with antiphosphotyrosine antibody after stimulation with EGF. Upon immunoprobing with antiphosphotyrosine antibody, several bands were observed. The band at 170 kDa, the size of the EGF-R, was markedly less phosphorylated in the DHT-treated fibroblasts (Fig. 2Go). However, this 170-kDa band does not necessarily represent just the EGF-R. The immunoprecipitation with the antiphosphotyrosine antibody was not specific for the EGF-R, because of the potential phosphorylation of other ErbB receptors and other proteins. Therefore, in further experiments we specifically immunoprecipitated with anti-EGF-R and immunoprobed with antiphosphotyrosine antibody. This revealed a single band at 170 kDa, which again was reduced in the DHT-pretreated condition, confirming that EGF-R phosphorylation was reduced in DHT-treated cells.



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  		Figure 2. The effect of chronic DHT exposure on EGF receptor phosphorylation in day 18 female mouse lung fibroblasts. Cells were treated with 100 ng/ml EGF for 2 min at 37 C, as indicated at the bottom of the blots. Lysates were immunoprecipitated (IP) with antiphosphotyrosine antibody ({alpha}-ptyr; upper blot) or anti-EGF-R antibody (lower blot) and immunoblotted with antiphosphotyrosine antibody.

 
TGFß-R-specific binding was also altered by in utero DHT exposure. Chronic intrauterine DHT exposure caused an increase in TGFß-R-specific binding (Fig. 3Go). Specific TGFß-R binding in female cells was increased by DHT exposure to 173 ± 39% of the control values (P < 0.05). Male cells treated with DHT also showed an increase to 280 ± 64% over that in control cells (P < 0.05). Although binding was increased relatively more in male cells, the difference in response to DHT treatment between the sexes was not statistically significant. As again expected, there was no significant difference in TGFß-R-specific binding between the sexes at this gestational age (female cells, 869 ± 133 cpm/nmol DNA; male cells, 659 ± 78 cpm/nmol DNA).



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  		Figure 3. The effect of chronic DHT (gray bars) exposure on specific [125I]TGFß-R binding in fetal day 18 mouse lung fibroblasts compared with experiment-specific controls (black bars). Females, n = 30; males, n = 30. Values are the mean ± SEM. *, P < 0.05.

 
Baseline cell proliferation in untreated control fetal lung fibroblasts was modestly higher in female than in male cells, but this was not statistically significant (female cells, 2962 ± 782 dpm/nmol DNA; male cells, 2335 ± 525 dpm/nmol DNA). TGFß1 induced thymidine incorporation in control cells (female cells, 4903 ± 1145 dpm/nmol DNA; male cells, 3894 ± 521 dpm/nmol DNA; P < 0.05), similar to results we have previously reported (27). DHT exposure also caused a significant increase over baseline thymidine incorporation in female fibroblasts (4187 ± 1055 dpm/nmol DNA; P < 0.05), and a mild decrease in male fibroblasts (1872 ± 436 dpm/nmol DNA). Furthermore, when the DHT-exposed female cells were treated with TGFß1, cell growth was significantly elevated to 231 ± 57% of that in controls treated with TGFß1 (6836 ± 1506 dpm/nmol DNA; P < 0.05; Fig. 4Go). However, male cells showed a slightly, but not statistically significant, decrease in TGFß1-induced proliferation after DHT treatment (controls, 3894 ± 521 dpm/nmol DNA; DHT, 1866 ± 334 dpm/nmol DNA).



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  		Figure 4. The effect of chronic DHT (gray bars) exposure on TGFß1-induced [3H]thymidine incorporation in fetal day 18 mouse lung fibroblasts compared with experiment-specific controls (black bars). Females, n = 30–31; males, n = 24–26. Values are the mean ± SEM. *, P < 0.05.

 
The effect of chronic intrauterine DHT exposure on type II cell maturation was studied by evaluating the expression of mRNA for surfactant proteins SP-B and SP-C. The cDNA for SP-B identified a single 1.6-kb mRNA transcript, and the cDNA for SP-C recognized a single 0.8-kb transcript as described by others (40). The baseline expression of both SP-B and SP-C was not significantly different between males and females (SP-B: females, 8.77 ± 3.7; males, 9.29 ± 2.8; SP-C: females, 7.9 ± 1.5; males, 12.2 ± 2.0), in agreement with previous studies (41). DHT exposure decreased both SP-B and SP-C mRNA expression (Fig. 5AGo), and the magnitude of decrease for each message was similar in both sexes (SP-B: female controls, 8.77 ± 3.7; female DHT, 5.91 ± 2.2; male controls, 9.29 ± 2.8; male DHT, 5.82 ± 3.1; SP-C: females, 7.9 ± 1.5; female DHT, 6.76 ± 1.6; males, 12.2 ± 2.0; male DHT, 8.9 ± 1.0). Data were combined for final analysis. Densitometry values are expressed as a percentage of the ratio of surfactant protein to RPL32 mRNA levels compared with the experiment-specific controls. DHT exposure decreased SP-B expression to 55 ± 0.1% of the control value (P < 0.05). SP-C expression in the DHT-exposed lungs was reduced to 76 ± 0.04% of the control values (P < 0.05; Fig. 5BGo).



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  		Figure 5. A, Representative Northern blot of the effect of chronic DHT exposure on expression of SP-B (upper blot) and SP-C mRNA (lower blot) in day 18 fetal female and male lungs. B, Ratio of densitometry of SP-B and SP-C to the internal standard RPL32, for DHT-treated lungs (gray bars; n = 9–11) compared with controls (black bars; n = 10–11). Results for females and males combined (n = 9–10 for SP-B and n = 11 for SP-C) are shown. Values are the mean ± SEM. *, P < 0.05.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
To better understand the mechanisms underlying the inhibitory effect of DHT on lung maturation, we treated animals with DHT throughout lung development to affect the normal progression in maturation. It is well known that the male disadvantage in lung maturation that is mediated via androgens leads to a reduction in choline incorporation into DSPC (5, 6, 7, 42). As lung maturation is mediated by a mesenchymal-epithelial cell communication process involving an unidentified mediator(s) (16), we focused in this study on regulation of the EGF and TGFß receptors in the mesenchyme to gain some insight into the signaling pathways affected by androgens. We used sex-specific fetal lung fibroblasts late in gestation when development of fibroblast-type II cell communication controlling epithelial cell maturation leading to surfactant synthesis is complete. Therefore, we did not expect significant sex-specific differences in the control animals, because the male cells at this gestational age have caught up in their maturation in preparation for birth (28, 29). We did observe small trends toward a more advanced developmental stage in the female, including an increase in cell growth, a slightly higher TGFß-R binding, and a slightly lower EGF binding as well as a marginal decrease in SP mRNA levels at baseline in the female lungs. This could be part of the preparation for postnatal life, where the already mature organ initiates growth and structural remodeling (26, 27, 29).

The DHT dose used was based on previous dose-response studies of DHT in utero administration (4) to alter lung maturation and on previous studies in which we administered DHT specifically to alter lung maturation in the fetal mouse (8). The dose-response studies showed that a dose of 1 mg/kg maternal BW lowered female surfactant production to male levels without affecting males, whereas 10 mg/kg had a small effect on males, and 50 mg/kg had a significant effect on males as well as females. The DHT dose we used for this study was in the higher range, because the goal of affecting both sexes was to be able to more conclusively evaluate how androgen alters basic mechanisms of lung development. Although the fetal androgen level is probably supraphysiological (4), the fact that both males and females are affected provides significant support for the conclusion that androgen affects mechanisms of cell growth and differentiation in the fetal lung. These studies extend observations on fetal lung growth and differentiation using experimental designs in which fetal lungs were exposed in vivo to unchanging but physiological androgen levels (6, 24, 31, 43). Importantly, they show that androgen does regulate basic mechanisms controlling cell growth and differentiation in both sexes.

The lung expresses androgen receptors and is, therefore, a candidate organ for direct androgen effects. Androgen receptors bind to DNA to up-regulate or down-regulate gene expression. It is likely that chronic DHT exposure alters the expression of genes that are ultimately involved in the direct control of lung cell differentiation. However, the signal transduction processes affected by the androgen-induced reduction of surfactant synthesis are unknown. Both EGF-R and TGFß-R mechanisms are involved in the control of lung maturation (12, 23, 24, 25). As both are affected by DHT exposure, our data suggest that androgen effects on lung maturation normally involve the regulation of both of these signaling mechanisms.

Our study found alterations of two regulators of positive and negative control of fetal lung maturation. First, we found that DHT exposure reduced EGF-R binding and EGF-R phosphorylation in fetal lung fibroblasts. This occurred at a time during gestation when EGF binding in the fetal lung fibroblast has normally reached a maximum (22), coincident with fibroblast induction of surfactant synthesis in the type II cells. This contrasts with our previous findings that acute in vitro DHT treatment of primary fetal lung fibroblast cultures from late gestation results in increased EGF binding and EGF-R protein expression (34) and indicates that the androgen delay of lung maturation involves effects on the maturational development of signals controlling cell differentiation. Chronic DHT exposure appears to prolong fibroblast immaturity in the developing lung by inhibiting the late gestation ontogenesis of the EGF-R. This mechanism might constitute one step in the process of androgen-mediated delay in surfactant synthesis. In normal male fetal development, androgen levels peak during lung immaturity, then decline as lung maturation is initiated (44, 45, 46).

Second, we found that TGFß-R binding, which normally reaches its nadir on day 18 in the fetal mouse lung in both sexes (27), was elevated on day 18 after DHT exposure. TGFß1 is produced by the immature fibroblast and has an inhibitory effect on type II cell maturation (23). As maturation of the fetal fibroblast progresses, TGFß-R activity is down-regulated at least in part by a decrease in TGFß-R binding (27). As TGFß is usually produced by immature fetal lung fibroblasts (23), we speculate that altered production might be an additional mechanism by which DHT exposure prolongs fibroblast immaturity through prolongation of TGFß-R activity.

Third, we found that TGFß-induced cell proliferation was increased in DHT-pretreated female cells compared with controls. Although early lung development is characterized by cell proliferation, late lung development is predominantly governed by cell differentiation processes during which proliferation is markedly reduced (43). This suggests that in addition to increased TGFß-R binding, there is prolongation by DHT of specific receptor-mediated events that promote proliferation. Although these events are unknown, we speculate that this involves increased amounts of signal transduction intermediates, which allow the proliferative response to TGFß to predominate. It is possible that the higher endogenous androgen level in the male fetus is the reason why enhanced proliferation after chronic DHT exposure was not also seen in male fibroblasts, although there was a similar effect on the EGF-R and TGFß-R as well as surfactant protein mRNA in both sexes. This is similar to our previous finding that when fetal mouse lung fibroblasts from an earlier gestational age were treated with DHT cell growth was inhibited in male cells and was promoted in female cells (27).

Fourth, DHT treatment significantly decreased surfactant protein B and C mRNA expression. An effect of DHT on surfactant protein gene expression has not been previously described. Our results of the androgen effect on surfactant protein mRNA expression show that androgen inhibits other aspects of type II cell maturation in addition to surfactant phospholipid synthesis. The stimulatory effects of EGF and the inhibitory effects of TGFß1 on SP-A expression have been described (12). We have previously shown that in late gestation fetal rat lung fibroblasts EGF-R overexpression followed by receptor activation had a stimulatory effect on type II cell DSPC synthesis but no effect on surfactant protein B and C mRNA expression (47). It is possible that the down-regulation of surfactant protein genes after chronic DHT exposure is mediated by direct or indirect effects on type II cells through the TGFß-R.

From our data we propose a model of lung development in which DHT inhibits the EGF-promoted fibroblast-type II cell communication and also promotes the inhibitory effect of TGFß1 on this cell-cell communication process (Fig. 6Go). Chronic intrauterine androgen exposure delayed the development of the EGF-R and TGFß-R as well as surfactant protein ontogenesis for approximately 1–2 gestational days compared with the findings of previous studies (22, 27, 34, 41). These results may explain the delay in surfactant synthesis seen in normal male fetal development.



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  		Figure 6. Proposed model of fibroblast-type II cell communication in late fetal lung development. Hormones and growth factors are involved in controlling the process. In particular, EGF promotes and TGFß1 inhibits the process of fibroblast-type II cell communication leading to surfactant synthesis. DHT inhibits positive regulation by EGF and stimulates negative regulation by TGFß1.

 


   Acknowledgments
 
We thank Drs. Olaf Dammann and Kermit L. Carraway III for helpful discussions and critical reading of the manuscript.


   Footnotes
 
1 This work was supported by NIH Grant HL-37930. Back

Received December 27, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Torday JS, Nielsen HC, Fencl M 1981 Sex differences in fetal lung maturation. Am Rev Respir Dis 123:205–208[Medline]
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