Hormonal regulation and cellular localization of fatty acid synthase in human fetal lung

Sameer Wagle1, Anh Bui1, Philip L. Ballard1, Henry Shuman2, John Gonzales1, and Linda W. Gonzales1

1 Department of Pediatrics, University of Pennsylvania, Children's Hospital of Philadelphia, and 2 Institute for Environmental Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Fatty acid synthase (FAS; EC 2.3.1.85) supplies de novo fatty acids for pulmonary surfactant synthesis, and FAS gene expression is both developmentally and hormonally regulated in the fetal lung. To further examine hormonal regulation of FAS mRNA and to determine the cellular localization of FAS gene expression, we cultured human fetal lungs (18-22 wk gestation) as explants for 1-4 days in the absence (control) or presence of glucocorticoid [dexamethasone (Dex), 10 nM] and/or cAMP agents (8-bromo-cAMP, 0.1 mM and IBMX, 0.1 mM). FAS protein content and activity increased similarly in the presence of Dex (109 and 83%, respectively) or cAMP (87 and 111%, respectively), and responses were additive in the presence of both hormones (230 and 203%, respectively). With a rabbit anti-rat FAS antibody, FAS immunoreactivity was not detected in preculture lung specimens but appeared in epithelial cells lining the tubules with time in culture. Dex and/or cAMP markedly increased staining of epithelial cells, identified as type II cells, whereas staining of mesenchymal fibroblasts was very low under all conditions. With in situ hybridization, FAS mRNA was found to be enriched in epithelial cells lining the alveolar spaces, and the reaction product increased in these cells when the explants were cultured with the hormones. The increased FAS mRNA content in the presence of Dex and/or cAMP is primarily due to increased stabilization of mRNA, although Dex alone increased the transcription rate by ~30%. We conclude that hormonal treatment of cultured human fetal lungs increases FAS gene expression primarily by increasing stability of the message. The induction of FAS during explant culture and by hormones occurs selectively in type II epithelial cells, consistent with the regulatory role of this enzyme in de novo synthesis of fatty acid substrate for surfactant synthesis in perinatal lungs.

surfactant synthesis; lung development; glucocorticoids; adenosine 3',5'-cyclic monophosphate


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

SUCCESSFUL TRANSITION to extrauterine life for mammals requires adequate pulmonary surfactant, the complex phospholipid and protein mixture that forms a monolayer at the air-liquid interface within the alveolus. Surfactant production in the fetus is under multihormonal regulation, but the precise mechanisms involved in hormonal influence and interactions are not yet fully defined (28, 33).

Toward the end of gestation, there is a surge in surfactant production, specifically dipalmitoylphosphatidylcholine, which is the predominant and surface-active lipid in surfactant as demonstrated by increased phosphatidylcholine (PC) content and choline incorporation into PC in several species (33) and increased tissue phospholipid in the human lung (37). Fatty acids are a major precursor of surfactant phospholipid (25), and fatty acid synthesis in fetal lungs increases sharply during late gestation in several species (15, 25, 35), although the relative contributions of fatty acids from the circulation versus de novo synthesis to the total surfactant phospholipid pool are unknown. Recent studies have shown a close temporal correlation between choline incorporation into PC and an increased rate of fatty acid synthesis in vivo (35) and during hormonal exposure of fetal lung explants in culture (19, 27, 29). Moreover, the activity of fatty acid synthase (FAS), the multifunctional enzyme catalyzing fatty acid synthesis, increases during late gestation in rat (30), rabbit (15), and human (37) fetal lungs and is stimulated by glucocorticoids (19, 27, 30, 31) and agents that increase cAMP (18) in explant culture. The combination of dexamethasone (Dex) and cAMP increases FAS mRNA content synergistically (~10-fold) (18). Both basal and glucocorticoid-stimulated FAS activities were greater in a cell population enriched in type II cells than in fibroblasts isolated from human fetal lung explants (19).

The present studies were undertaken to evaluate the relative content of FAS protein and mRNA in different cell types of the human fetal lung in situ and to identify hormonally responsive cell types. Hormonal effects on FAS protein content of lung explants were quantitated to investigate the discrepancy between induction of mRNA and FAS activity. To investigate glucocorticoid and cAMP mechanisms of action on FAS expression in the human lung, transcription rate and message stability were compared in control and hormone-treated explants.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Materials

Dex, 8-bromo-cAMP, malonyl-CoA, acetyl-CoA, and other biochemicals were purchased from Sigma (St. Louis, MO). [2-14C]malonyl-CoA (41.1 mCi/mmol), [3H]uridine (39.6 Ci/mmol), [32P]dCTP (3,000 Ci/mmol), and [32P]UTP (3,000 Ci/mmol) were purchased from NEN (Boston, MA). 35S-UTP (1,500 Ci/mmol) was purchased from Amersham (Arlington Heights, IL). The riboprobes were prepared with the Gemini T3/T7 RNA polymerase kit (Promega, Madison, WI). Waymouth MB-752/1 medium was obtained from the Cell Culture Facility (University of Pennsylvania, Philadelphia) and supplemented with penicillin (100 U/ml), streptomycin (100 µg/ml), and Fungizone (2.5 µg/ml). The human mammary adenocarcinoma cell line SkBr3 was obtained from the American Type Culture Collection (Manassas, VA). Enhanced chemiluminescence (ECL) reagent kit was purchased from Amersham.

Explant Culture

Human fetal lungs were obtained from 18- to 22-wk-gestation therapeutic abortions under protocols approved by the Committee on Human Research at The Children's Hospital of Philadelphia. Fetal lung parenchyma was minced into 1-mm3 pieces and placed in organ culture as previously described (17). Briefly, tissue pieces were distributed in two parallel strips on 60-mm culture dishes placed on a platform that rocked (3 oscillations/min) to expose the explants alternately to serum-free Waymouth medium (2 ml/dish) or an atmosphere of 95% air-5% CO2. The explants were maintained for 1-5 days in medium without (control) or with Dex (10 nM), 8-bromo-cAMP (0.1 mM) plus IBMX (0.1 mM), or both Dex and the cAMP agents. IBMX was added to all explants treated with 8-bromo-cAMP to maintain tissue cAMP levels. Fresh medium was added every 24 h. Tissue explants were harvested either before culture (preculture) or at various times during culture. Unless otherwise stated, hormones were added to the medium of treated explants after 24 h of culture. The hormone concentrations used maximally stimulated PC synthesis from a previous study by Gonzales et al. (17; also Gonzales, unpublished observations).

SkBr3 cells were grown in DMEM-10% fetal calf serum supplemented with insulin (1 µg/ml) as previously described (40).

SDS-PAGE and Western Blot

Sonicates of fetal lung explants were assayed for total protein with the method of Bradford (9) with a Bio-Rad protein assay kit (Bio-Rad, Richmond, VA), and aliquots were subjected to SDS-PAGE with Tris-glycine buffer and 5% acrylamide gels according to the method of Laemmli (24). In some experiments, the sonicates were prepared in a protease inhibitor mixture containing benzamidine (1 mM), N-ethylmaleimide (5 mM), and phenylmethylsulfonyl fluoride (40 mM). The proteins were then transferred electrophoretically overnight to nitrocellulose. After transfer and blocking with 5% milk, immunoblotting was performed with the primary antibody, polyclonal rabbit anti-rat FAS (1:3,000 dilution), and the secondary antibody, goat anti-rabbit IgG conjugated with horseradish peroxidase (1:10,000 dilution), as previously described (6). Monospecific polyclonal antisera directed against rat mammary gland FAS was produced in rabbits and further processed to a partially purified IgG fraction (generously donated by Dr. Stuart Smith, Children's Hospital Medical Center, Oakland, CA). The antibody recognizes both rat FAS and the human SkBr3 cell line (40). ECL Western blotting detection reagents were used to expose Kodak-X-OMAT AR film as per the manufacturer's instruction (Amersham International). Exposure times used gave a linear signal between 25 and 200 µg of protein applied. Developed films were scanned with a densitometer (Hoefer Scientific, San Francisco, CA), and relative densities of peak area were calculated (in optical density units) with the GS370 program. Extracts prepared by sonication in the presence and absence of proteinase inhibitors gave identical band pattern and intensity.

Immunostaining

The experiments used four human lungs of 18-22 wk gestation, which were examined before and after 1-4 days of explant culture. Lung explants were fixed (2 h) with cold 4% paraformaldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) and then washed in sodium cacodylate buffer for 1 h. The tissue was cryoprotected in 10% sucrose in 0.1 M sodium cacodylate for 1 h, 20% sucrose in the same buffer for 1 h, and 30% sucrose in the same buffer overnight at 4°C. The tissue was mounted with polyfreeze tissue-freezing medium (optimum cutting temperature compound) cooled with dry ice and 2-methylbutane (Sigma). Cryostat sections 5 µm thick were cut at -20°C, placed on gelatin-chrom-alum-coated slides, and air-dried.

Tissue sections were immunostained according to Williams et al. (43), modified as described (8) with two preincubations in 0.1% sodium borohydride in PBS for 5 min to reduce endogenous fluorescence. Staining was done by sequential incubation of sections with rabbit anti-FAS diluted 1:100 in PBS containing 0.3% Triton X-100, 3% bovine serum albumin, and 5% goat serum followed by incubation in Texas Red-conjugated goat anti-rabbit IgG (Organon Teknika, Durham, NC) diluted 1:1,000 in the same buffer solution. After extensive washes in PBS with 0.3% Triton X-100 and then PBS alone, the sections were air-dried and the coverslips were sealed with Mowiol (Calbiochem, San Diego, CA). Nearby sections were stained with hematoxylin and eosin as a reference guide for tissue morphology.

For colocalization studies, adjacent sections were sequentially incubated with either rabbit anti-rat FAS antibody or rabbit anti-human surfactant proprotein C (anti-NPROSP-C; 1:300) and then goat anti rabbit IgG indocarbocyanine conjugated, followed by rabbit anti-human SP-B (1:500) directly conjugated with fluorescein.

For controls, rabbit preimmune serum (diluted 1:100) was substituted for the primary antibody. All sections were examined with conventional fluorescent microscopy. Texas Red (and indocarbocyanine) fluorescence were detected at wavelengths of 563 nm (excitation) and 625 nm (emission), and fluorescein was observed at 505 and 535 nm, respectively. Photographs in each individual figure (Figs. 3-6) were made with the same exposure for all specimens.

FAS Assay

The activity of FAS was determined in supernatant fractions (15,600 g for 15 min) of tissue by radioactive malonyl- CoA utilization for total fatty acid synthesis with the method of Roncari (32), and enzyme activity is expressed as nanomoles of malonyl-CoA utilized per milligram of protein (9) per minute as described previously (19). Under the conditions used, activity was proportional to the protein added and incubation time.

mRNA Analysis

To extract total RNA, explant tissue (50-250 mg wet wt) was sonicated in acidic guanidinium thiocyanate with the method of Chomczynski and Sacchi (13). RNA was quantified by absorbance at 260 nm, and purity was determined by the ratio of absorbance at 260 nm to that at 280 nm. Specific amounts (0.5-4.0 µg) of total RNA extract were applied to nitrocellulose filters (Duralose-UV, Stratagene) for dot blot analysis and mRNA quantitation, as previously described (4), by hybridization to a 32P-labeled probe prepared by random-primer labeling with [32P]CTP and the Ready-To-Go kit (Pharmacia) with a human FAS cDNA probe (clone Pg8). This cDNA contains a 2-kb fragment complementary to a sequence near the 3'-end of the FAS coding region (generous gift from Dr. D. Chalbos, INSERM, U148, Montpellier, France). The Pg8 cDNA was isolated by differential screening of a progestin-treated human breast epithelial cell line (MCF7) (11). The human beta -actin cDNA was kindly supplied by T. White and B. Benson (California Biotech, Mountain View, CA).

Nuclear Transcription Elongation (Run-On) Assay

Transcription was assayed as previously described (42). Briefly, nuclei were prepared from explants (~100 mg of tissue in duplicate) by homogenization and centrifugation. Nuclei (2.5 × 107) were resuspended in reaction buffer and incubated with 0.5 mM ATP, 0.25 mM CTP, 0.25 mM GTP, 100 µCi of [32P]UTP (3,000 Ci/mmol), and 40 U of RNase inhibitor at 37°C for 30 min. The nuclei were then successively digested with RNase-free DNase I and proteinase K and passed through gel-filtration (Sephadex G-50) spin columns to remove the free nucleotides.

Unlabeled cDNA probes in the Bluescript plasmid were linearized, denatured, and applied to a nitrocellulose filter strip (5 µg/dot) in a dot blot apparatus. Each filter strip contained cDNAs for FAS, SP-B, and human beta -actin as well as for the Bluescript plasmid without insert. Ballard et al. (4) previously found that beta -actin mRNA content remains constant during the second trimester and during explant culture with and without dexamethasone. The filters were hybridized with 2.5 × 106 counts/min (cpm) of labeled RNA at 42°C for 3-4 days and washed under high-stringency conditions, and single-stranded RNA was removed by incubation with RNase A. The filters were exposed to film at -70°C for 2-10 days, and the autoradiograms were quantified by densitometric scanning with exposures within the linear range for the film (units are arbitrary densitometric units for counts per minute in FAS mRNA per 2.5 × 106 cpm in total RNA). Data were corrected for the beta -actin control when a beta -actin experimental point varied >10% from others in the experiment.

Pulse-Chase Assay for mRNA Stability

Explants were labeled (1 ml medium/dish) on day 4 of culture with 500 µCi of [3H]uridine (specific activity 45 Ci/mmol) for 16 h, then washed and incubated in medium containing cold uridine and cytidine (5 mM each; equals time 0). Tissue was harvested at various time points thereafter and washed three times in fresh medium to remove unincorporated label, and total RNA was isolated.

Bluescript plasmids containing FAS, beta -actin, or no cDNA insert were linearized with EcoR I (FAS) or Kpn I (1 U/µg plasmid), extracted, and prepared as previously described (42). Four to six micrograms of the linearized DNA were added to each well of a dot blot apparatus, rinsed with 10× saline-sodium citrate (SSC; 1× SSC is 0.15 M NaCl and 0.015 M sodium citrate, pH 7.0), and the nitrocellulose filter was dried at 80°C in a vacuum oven for 2 h. Disks corresponding to each well were cut from the filter with a biopsy punch and stored at room temperature until hybridization with labeled RNA.

To hybridize the filter disks, each of which contained a single type of cDNA insert or empty plasmid (background control), one of each type was placed together in a microfuge tube with 75 µl of a preannealing solution and incubated at 42°C for 4 h. Meanwhile, the labeled RNA was boiled and quenched on ice, and 15 × 106 cpm were added to the disks and hybridized at 42°C for 48 h. The disks were washed twice for 30 min in 0.2× SSC with 0.1% SDS and twice for 15 min in 2× SSC, all at 50°C, and incubated in 2× SSC containing RNase A (10 µg/ml) at 37°C for 30 min followed by two washes in 2× SSC for 15 min at room temperature. The disks were separated, and the RNA was dissolved in 0.4 N NaOH, neutralized with acetic acid, and counted by scintillation counting.

In Situ Hybridization

Hybridization studies were performed with two lungs of 19 and 22 wk gestation. Tissue was harvested after 3 days of explant culture with and without hormones, then fixed for 1 h at 4°C in 4% paraformaldehyde in PBS (pH 7.2). The tissue was then incubated overnight in 30% sucrose in PBS, embedded in optimum cutting temperature compound (Tissue Tek), quick-frozen in liquid nitrogen, and stored at -70°C until sectioned.

Probe preparation. A 751-bp fragment was digested from the 2-kb human FAS cDNA (clone Pg8) with Sac I and BamH I and ligated into corresponding sites of pBluescript KS. The orientation was confirmed by sequencing with both the dideoxynucleotide method (Sequenase version 2.0 kit, US Biochemical, Cleveland, OH) and restriction digestions and was found to have ~83% homology with rat FAS cDNA (bases 5730-6570) (2). pBluescript containing the 751-bp cDNA was linearized with Nco I near the center of the insert (to obtain transcribed probes of smaller size) and was used as a template for the preparation of cRNA probes with Riboprobe System Gemini-T3/T7 (Promega). Transcriptions were performed according to the manufacturer's specifications in the presence of 300 µCi of uridine 5'-[35S]thiotriphosphate. Radiolabeled transcripts of ~370 bp each for both the sense and antisense probes were then purified by size-exclusion chromatography through small Sephadex G-50 columns (Boehringer Mannheim), and purity was confirmed by running on denaturing 5% Long Ranger (AT Biochem) sequencing gels. When tested for hybridization to Northern blots containing RNA from explant tissue, the T7-primed probe (antisense) hybridized to an ~9-kb band, whereas the T3-primed probe (sense) did not. Riboprobe orientations were confirmed by restriction analysis of the plasmid.

Hybridization. In situ hybridization was carried out as previously described (7). Sections warmed to room temperature and air-dried were fixed with 3% paraformaldehyde in PBS, rinsed with PBS, treated for 10 min at room temperature with proteinase K (10 µg/ml), and rinsed twice with 2× SSC and then with 0.1 M triethanolamine (pH 8.0) containing 0.25% acetic anhydride for 10 min. The sections were rinsed with 2× SSC for 1 min, rinsed with 0.1 M PBS, treated with 0.1 M Tris-glycine (pH 7.0) for 30 min, rinsed with 2× SSC for 1 min, and then dehydrated with graded ethanol and air-dried. The sections were hybridized overnight at 50°C with 2 ng of DNA (~5 × 105 cpm) and rinsed with 2× SSC at room temperature, then with 50% formamide in 2× SSC at 52°C. The sections were digested at 37°C with RNase A (100 µg/ml 2× SSC) for 30 min and then rinsed further with 2× SSC and 50% formamide in 2× SSC, followed by overnight incubation with 0.05% Triton X-100 in 2× SSC. A quick ammonium acetate rinse the next day was followed by dehydration in graded ethanol and xylene. After dehydration, the slides were dipped in Kodak NTB2 photographic emulsion for autoradiography, allowed to expose for 14-21 days at 4°C, and then developed, counterstained with hematoxylin and eosin, and photographed. Tissues from all experimental conditions were processed together and exposed for the same time.

Statistics

All data were analyzed by ANOVA, and comparisons of multiple groups were made with Fisher's protected least-squares difference test. The level of significance was P < 0.05.


    RESULTS
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INTRODUCTION
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Effect of Hormone Treatment on FAS Protein Content

Previous studies (19) established hormonal regulation of FAS activity in cultured human fetal lungs, but no information is available on the content of FAS protein. To address the question of whether a quantitative change in FAS protein correlated directly with the change in FAS activity, the hormonal effects on FAS protein were determined by Western analysis. Immunoreactive FAS in sonicates of cultured lung explants was present as a single band of ~236 kDa by SDS-PAGE and immunoblotting (Fig. 1). The intensity of the ECL signal was linear from 25 to 250 µg of total protein loaded (data not shown). The sensitivity and useful range of the FAS antibody were determined by utilizing protein from SkBr3 cells, a human mammary carcinoma cell line known to be exceptionally rich in FAS [~24% of total cell protein as FAS (40)]. Sonicates of preculture lung tissue (FAS band intensity below detectable level) were spiked with 0.05-30 µg of protein from SkBr3 cells, and Western analysis was performed. The antibody binding dose-response curve was linear (densitometry not shown) over the entire range, with a calculated sensitivity of <= 240 ng FAS.


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Fig. 1.   Effect of glucocorticoid and cAMP on fatty acid synthase (FAS) protein content in explants cultured for 5 days. Explants from 2 lungs (lung 1, 21 wk gestation, left 4 lanes; lung 2, 22 wk gestation, right 4 lanes) were cultured in absence of hormones [control (C)] or in presence of dexamthasone [Dex (D); 10 nM], 8-bromo-cAMP (8-BrcAMP; 0.1 mM) plus IBMX [0.1 mM; cAMP agents (cA)], or D+cA, and 200 µg total protein/lane were applied to 5% SDS-PAGE gels. Western blots were treated with rabbit anti-rat FAS (IgG; 1:3,000) and horseradish peroxidase-conjugated secondary (1:10,000) antibodies and enhanced chemiluminescence (ECL) reagent. No. on left, molecular mass in kDa.

Both Dex (10 nM) and cAMP (0.1 mM) increased the amount of FAS protein in cytosols extracted from explants on day 5, and the increase was additive with combined hormone treatment (Fig. 1). With Western analysis, control levels of FAS protein were low and variable between lungs, but the relative hormonal responsiveness was similar. Densitometric scanning showed that the FAS protein content increased 109% above the control level in the Dex-treated, 87% in the cAMP-treated, and 230% in the Dex plus cAMP-treated explants (Table 1). In the same experiments, FAS activity was significantly stimulated by hormonal treatments as previously described (19), and the magnitude of the increases was comparable to that for FAS protein content (Table 1). Moreover, Western analysis of additional lungs (7 lungs total) cultured with both agents (Dex plus cAMP) confirmed the magnitude of increases in the FAS protein content (327 ± 38% of control value) as well as in FAS activity (238 ± 72%) and FAS mRNA content (1,042 ± 116%; P < 0.05 vs. control lungs for all values).

                              
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Table 1.   Effect of Dex and cAMP on FAS protein content, activity, and mRNA content

Effect of Hormone Treatment on FAS mRNA Content

Gonzales et al. (18) previously demonstrated a 9-kb FAS mRNA band by Northern analysis of cultured human lungs and found that the signal was increased by both Dex and cAMP treatment. To directly compare the hormonal effects on FAS protein and activity with mRNA content, in the present study, each of these parameters was measured in the same explants treated with optimal hormone concentrations (Table 1). With Dex or cAMP alone, the increase in FAS mRNA content was comparable to the increases in FAS activity and protein. In contrast, simultaneous treatment with both hormones increased FAS mRNA content synergistically (~7-fold) compared with additive (2-fold) increases in FAS protein content and activity. There were no new bands detected in the presence of hormones, and similar increases were measured on the 9-kb band by Northern analysis (18).

Effect of Hormones on FAS Transcription Rate

Culture for 3-5 days has previously been shown to increase the FAS mRNA content of human fetal lung explants in the absence as well as in the presence of Dex (18). In the present study, explants cultured in the absence of hormones had a higher transcription rate of FAS mRNA on days 3 and 5 of culture compared with that on day 1 of culture (1.56 ± 0.11- and 1.74 ± 0.23-fold vs. day 1, respectively; P < 0.05; n = 3 lungs, with 2-3 determinations/lung). Earlier experiments also showed increased FAS mRNA content of explants exposed to hormones for 24 h on day 4 of culture (18). Thus, in the present experiments, this relatively short exposure protocol was used to examine primary hormonal effects on transcription. Exposure to Dex (24 h on day 4), increased the FAS gene transcription rate compared with control conditions, whereas cAMP alone had no effect (Table 2). Exposure to both Dex and cAMP significantly increased transcription compared with that with Dex alone. In the same experiments, there was no hormonal effect on the transcription rate of the beta -actin gene, and transcription of the SP-B gene was increased by both Dex and cAMP (data not shown) as previously reported (42).

                              
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Table 2.   Effect of Dex and cAMP on transcription rate of FAS mRNA

Effect of Hormones on Stability of FAS mRNA

Because the increase in transcription rate in the presence of hormones did not appear to account for the total increase in FAS mRNA content, the effects of Dex and cAMP on the half-life of the mRNA were studied. In initial studies, explants were cultured 4 days in the absence (control) or presence of Dex plus cAMP, and then either actinomycin D (5 µg/ml) or 5,6-dichloro-1-beta -ribofuranosylbenzimidazole (60 mM) was added to inhibit RNA synthesis, and explants were harvested 4-20 h later. FAS mRNA degradation rates were similar (half-life = 15-18 h) in control and hormone-treated explants in the presence of either inhibitor. Because of the potential secondary action of these inhibitors on the synthesis of molecules involved in the turnover of FAS mRNA, these studies were considered inconclusive. Thus we carried out studies using a label-chase approach to examine stability of FAS mRNA under the different hormonal conditions. Explants were labeled with [3H]uridine at a high specific activity for 16 h (label), then "chased" with a high concentration of unlabeled uridine plus cytidine and harvested at intervals from 4 to 12 h (Fig. 2). Under these conditions, the presence of either Dex or cAMP significantly increased the half-life of FAS mRNA ~2-fold, and treatment with Dex plus cAMP increased the half-life ~4-fold (Table 3). As shown in a representative experiment (Fig. 2), the half-life of beta -actin mRNA, which was determined simultaneously in each of the experiments, did not change with hormonal conditions. The specific activity of total RNA (in cpm/µg RNA) was similar for control and hormone-treated explants at all time points.


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Fig. 2.   Effect of hormones on half-lives of FAS and beta -actin mRNAs. Explants were cultured in absence (control) and presence of Dex (10 nM), 8-BrcAMP (1 mM) plus IBMX (0.1 mM), or both Dex and cAMP agents. On day 5, explants were pulsed with [3H]uridine overnight and then washed to remove radiolabeled medium, and aliquots were harvested at 4-, 8-, and 12-h intervals thereafter. cpm, Counts/min. Values are means ± range of triplicate determinations from a representative experiment. A: half-life of FAS mRNA was greater in Dex- or cAMP-treated explants compared with control explants and even greater in Dex+cAMP-treated explants compared with either alone. B: half-life of beta -actin mRNA was similar in explants cultured under all conditions (same RNA samples as in A).


                              
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Table 3.   Half-lives of FAS and beta -actin mRNAs in presence of Dex and/or cAMP

Cellular Localization of FAS Gene Expression

Immunofluorescent localization of FAS. Immunoreactive FAS was below a detectable level in preculture fetal lungs of 18-22 wk gestation (Fig. 3A). However, by day 1 in culture, immunoreactive FAS was detectable (Fig. 3B), and the signal increased markedly during culture in the absence of serum or hormones. Immunoreactivity was most prominent in epithelial cells lining the tubules (Fig. 3, B and C, arrows) and increased between day 1 (Fig. 3B) and day 3 (Fig. 3C) in culture. There was no detectable specific immunofluorescence in mesenchymal cells of preculture lungs and only low levels in cultured tissue.


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Fig. 3.   Immunofluorescent localization of FAS protein in human fetal lungs of 21 wk gestation. A: preculture. B: day 1 in culture. C: day 3 in culture. D: nonimmune rabbit serum control of day 3 explants. Explants were cultured in absence of serum or hormones for 1-3 days. Explants were fixed in 4% paraformaldehyde and cryoprotected as described in METHODS, and 5 sections were exposed to primary (rabbit anti-rat FAS IgG; 1:100) and then secondary (goat anti-rabbit IgG Texas Red-conjugated) antibodies. Arrows, epithelial cells lining tubules. Bars, 12 µm.

Treatment of explants with Dex for 4 days (days 1-5 in culture) substantially increased immunoreactive FAS in epithelial cells (Fig. 4B), producing intense staining of the air space-lining cell layer. Staining over the mesenchyme remained minimal, resembling background staining seen in the preimmune serum control cells (Fig. 4E). In explants cultured in cAMP alone (0.1 mM; data not shown), fluorescence intensity similar to that of Dex-treated explants was observed and was also localized only in the epithelial cells. The intensity of staining in the explants treated with Dex plus cAMP (data not shown) appeared only slightly greater than that in explants treated with either hormone alone, probably reflecting the limitations of the quantitative aspects of the method. Similar hormonal effects were observed with three additional lungs (18, 21, and 22 wk gestation). The apparently lower FAS content of explants cultured 5 days without hormones (Fig. 4A) compared with day 3 control explants (Fig. 3C) was due to different film exposure times for the two experiments.


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Fig. 4.   Effect of glucocorticoid on FAS protein content in human fetal lung explants. Explants were cultured for 5 days in absence (A) and presence of 10 nM Dex (B), fixed, sectioned, and exposed to primary (rabbit anti-rat FAS IgG, 1:100) and then secondary (goat anti-rabbit IgG Texas Red-conjugated) antibodies. B, open arrow: epithelial cells showing increased fluorescence in presence of Dex. Sections from Dex-treated explants were also exposed to preimmune rabbit serum (in place of primary antibody) for specificity of fluorescence (E). Sections of control (C) and Dex-treated (D) explants were also stained with hematoxylin and eosin as a morphological reference to allow identification of the epithelial cells lining air spaces (solid arrows). Bars, 12 µm.

The FAS-positive cells were identified as type II epithelial cells by colocalization of both FAS and SP-C immunoreactivity with anti-SP-B immunostaining in separate but adjacent tissue sections. In each section tested, all FAS-immunoreactive cells (Fig. 5A) were also positive for SP-B (Fig. 5B), and all SP-C-immunoreactive cells (Fig. 5E) likewise were positive for SP-B (Fig. 5F), demonstrating colocalization of FAS and SP-C, the type II cell-specific marker (44).


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Fig. 5.   Colocalization of either FAS or surfactant protein (SP) C with SP-B in type II epithelial cells of human fetal lung (22 wk gestation) by fluorescence immunocytochemistry. Explants were cultured for 5 days in Dex plus cAMP, and then sections were immunostained with anti-rat FAS detected by goat anti-rabbit IgG indocarbocyanine (Cy3)-conjugated antibody (A) followed by anti-SP-B fluorescein-tagged antibody (B). L, lumen; M, mesenchyme. Thick open arrows, epithelial cells staining similarly for FAS and SP-B. C: phase-contrast image of this field. Adjacent sections were immunostained with anti-surfactant proprotein C (anti-NPROSP-C) antibody detected by goat anti-rabbit IgG Cy3-conjugated antibody (E), followed by anti-SP-B fluorescein-tagged antibody (F). Thin open arrows, epithelial cells staining similarly for SP-C and SP-B. G: phase-contrast image of this field. D and H: background fluorescence for Cy3 (nonimmune rabbit IgG) and fluorescein (nonimmune rabbit IgG fluorescein-tagged) antibodies, respectively. Bar, 12 µm.

In situ hybridization. In control explants, FAS mRNA, detected with the antisense probe (T7 primed), was enriched in epithelial cells surrounding the air spaces (Fig. 6A) and grain intensity was increased in these cells after cAMP (Fig. 6B) or Dex treatment (data not shown). Intensity of the signal was greatest for explants cultured in Dex plus cAMP (Fig. 6C). The sense probe (T3 primed) gave a much reduced signal (Fig. 6D) for explants cultured in Dex plus cAMP. Thus hormonal stimulation of the FAS mRNA transcripts was concentrated in the epithelial cells lining the air spaces.


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Fig. 6.   Localization of FAS mRNA in cultured human fetal lung (19 wk gestation) by in situ hybridization. Explants cultured for 5 days in absence (control; A) and presence of cAMP agents (B) or Dex (10 nM) plus cAMP (C and D) were hybridized with 35S-labeled oligonucleotide antisense (A-C) or sense cRNA (D). Results shown are bright-field photomicrographs, with arrows denoting specific hybridization signal located over epithelial lining cells. Similar results were obtained with a 22-wk lung. Bar, 12 µm.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

FAS is a widely distributed enzyme, essential for de novo synthesis of fatty acid, and although present in many cell types (10), it is more highly concentrated in tissues with high rates of lipid synthesis and is regulated by both dietary and hormonal factors. Fatty acid biosynthesis, FAS activity, and surfactant production are temporally correlated during lung development (27, 35). In addition to the important role of FAS in the production of fatty acid substrate for pulmonary phospholipid synthesis (34), it supplies the stimulatory lipid cofactor for choline-phosphate cytidylyltransferase (36, 46), a putative rate-regulatory enzyme in PC synthesis (20, 23, 38).

Hormonal regulation of FAS expression varies with species (47), tissue type, and state of tissue differentiation (14, 21). A previous study in our laboratory with human fetal lungs (19) and studies by others using rat (16, 30, 47), and rabbit (27) lungs in culture have demonstrated increases in either FAS activity or immunoreactive FAS protein content after treatment with glucocorticoids. In this study, we establish that cAMP, as well as glucocorticoids, causes equivalent and parallel increases in FAS protein content and FAS enzymatic activity, suggesting the absence of posttranslational modifications. Furthermore, we report the new observation that hormonal responsiveness of FAS is restricted to type II epithelial cells of cultured human fetal lungs.

FAS protein concentration has been examined in homogenates of developing fetal rat and rabbit lungs (15, 31) but has not been previously localized to a cell type(s) in situ in the lung. Batenburg et al. (5) reported a twofold increase in FAS mRNA after exposure of fetal rat type II cells to conditioned medium from cortisol-treated fibroblasts. The specific activity and increase in stimulation of FAS in type II cells isolated from human fetal lung explants exposed to Dex is also greater than that in fibroblasts from these explants (19). Moreover, the fatty acid biosynthetic rate is high in type II cells compared with that in whole lungs from adult rabbits (26).

We found that both the relative number of FAS-positive cells and intensity of immunostaining increased during culture without serum or hormones. This responsiveness was localized to the epithelial cells lining tubules, which were previously identified and characterized as type II cells with electron microscopy (12, 17). The increase in FAS content during culture may reflect, in part, the prostaglandin-mediated increase in endogenous cAMP previously described (3), which is likely responsible, at least in part, for the increases in PC synthesis and surfactant proteins. Endogenous cAMP likely increases FAS expression via stabilization of FAS mRNA, and another factor(s) may account for the observed increase in FAS transcription between day 1 and days 3-5 of culture. We detected no visible immunoreactive FAS in preculture tissue (18-22 wk gestation), consistent with the very low assayable FAS activity (19). This probably reflects low fatty acid synthesis in vivo at this gestational age, although some loss of FAS expression due to protein turnover during the ~24 h for shipping of the tissue cannot be ruled out.

Treatment of explants with Dex, cAMP, or both hormones further increased immunoreactive FAS in cells (type II) lining the air spaces, with no apparent effect on mesenchymal cells. We could not visually distinguish differences between the cellular levels of immunostaining after different hormonal treatments. Thus the increased tissue content of FAS after hormonal treatment appears to result from increased expression in individual epithelial cells. This is the first direct in situ evidence to support the role of type II cell FAS in mediating the glucocorticoid- and cAMP-induced maturation of surfactant phospholipid synthesis in fetal lungs. The quantitative contribution of fatty acid synthesized de novo versus other potential fatty acid sources such as triglyceride pools in adjacent fibroblasts (41) to total phospholipid synthesis will require further study.

Molecular mechanisms for developmental and hormonally mediated changes in FAS gene expression have not been fully determined in the fetal lung. We report the new finding that combined Dex and cAMP treatment produces a synergistic increase in FAS mRNA content. Our studies indicate that increases in both transcription and mRNA stability mediate the hormonal induction of increased FAS mRNA content in cultured human fetal lungs. The stimulatory effect of glucocorticoid is mediated via both processes, whereas cAMP exerts its individual effect solely by increasing stability but further enhances both the transcriptional rate and stabilization induced by glucocorticoid. These results agree with and directly substantiate findings from an inhibitor study reported earlier (18). Although the quantitative contributions of transcriptional and posttranscriptional regulatory events in FAS induction have not been rigorously studied, other studies (45, 47) provide evidence for operation of both processes in the fetal rat lung. In our inhibitor studies, the half-life of FAS mRNA in both control and hormone-treated explants was 15-18 h, comparable to values obtained for treated tissue by label-chase. This suggests that actinomycin D or 5,6-dichloro-1-beta -ribofuranosylbenzimidazole increases the half-life in control tissue, perhaps by inhibiting RNases or other degradative factors. The greater magnitude of transcriptional increase by glucocorticoid in rat lungs (46) suggests that the balance betweeen transcriptional and stability effects may be species dependent. Demonstration of a hormonally induced increase in FAS mRNA transcripts in type II epithelial cells by in situ hybridization establishes that increased FAS gene expression in these cells accounts for the increased FAS protein content. These findings suggest posttranscriptional effects of the hormone combination in addition to transcriptional increases in FAS mRNA content. Possibilities include different mRNA splicing, producing start sites with different translational efficiencies, protein degradation exceeding the increased rate of synthesis, compartmentalization of some mRNA, and limited translational machinery. Pulse-chase immunoprecipitation studies would aid in elucidating these mechanisms.

Recent cloning and sequencing of the rat FAS gene has identified potential regulatory hormone response elements for both glucocorticoid and cAMP within the gene (1). A sequence in the first intron is similar to the consensus glucocorticoid response element. However, in transgenic mice, a 2.1-kb region of the 5'-flanking FAS promoter sequence linked to the chloramphenicol acetyltransferase reporter gene conferred glucocorticoid responsiveness in lungs (39). Although no consensus cAMP response element sites have been identified, the 5'-flanking region contains eight sequences similar to the activator protein-2 sequence that may be involved in mediation of the cAMP response (1). The human FAS gene has also recently been sequenced, with confirmation of similar regulatory elements identified in the promoter or 5'-coding region (22). The differential responsiveness of epithelial cells versus fibroblasts may reflect different cohorts of transcription factors in these cell types.

In summary, our studies indicate that glucocorticoid and cAMP regulate expression of the FAS gene in type II epithelial cells of human fetal lungs, resulting in equivalent increases in FAS protein concentration and FAS activity. These findings further confirm the hormonal responsiveness of epithelial cells in the developing lung and their potential capacity for responding to an altered hormonal milieu by increasing the fatty acid substrate supply for de novo synthesis of surfactant phospholipid.


    ACKNOWLEDGEMENTS

We gratefully thank Kathy Notarfrancesco and Sreedevi Angampalli for expert technical skill in sectioning tissues and carrying out immunofluorescence and photomicroscopy, S. Smith (Oakland, CA) for generously donating the anti-rat fatty acid synthase (FAS) antibody, M. Beers (University of Pennsylvania, Philadelphia, PA) for donating the anti-human surfactant proprotein C (anti-NPROSP-C) antibody, S. Guttentag for donating the anti-human surfactant protein B fluorescein-tagged antibody, D. Chalbos (Montpellier, France) for generously donating the human FAS cDNA, and Eileen McAvoy for expert assistance in preparation of the manuscript.


    FOOTNOTES

This study was supported by National Heart, Lung, and Blood Institute Grant HL-19737 and an Endowed Chair in Neonatology.

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: L. W. Gonzales, Div. of Neonatology, Children's Hospital of Philadelphia, Abramson Pediatric Research Center, Rm. 414, 34th and Civic Center Blvd., Philadelphia, PA 19104 (E-mail: GonzalesL{at}EMAILCHOP.EDU).

Received 18 August 1998; accepted in final form 29 March 1999.


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