TGF-beta 1 inhibits surfactant component expression and epithelial cell maturation in cultured human fetal lung

Michael F. Beers1, Kola O. Solarin2, Susan H. Guttentag3, Joel Rosenbloom4, Annapurna Kormilli3, Linda W. Gonzales3, and Philip L. Ballard3

1 Pulmonary and Critical Care Division, Department of Medicine, University of Pennsylvania School of Medicine; 3 Division of Neonatology, Department of Pediatrics, Children's Hospital of Philadelphia; 4 Department of Anatomy and Histology, University of Pennsylvania School of Dental Medicine, Philadelphia 19104; and 2 Division of Neonatology, Department of Pediatrics, Allegheny University School of Medicine, Philadelphia, Pennsylvania 19134

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Transforming growth factor-beta 1 (TGF-beta 1) is a multifunctional cytokine shown to play a critical role in organ morphogenesis, development, growth regulation, cellular differentiation, gene expression, and tissue remodeling after injury. We examined the effect of exogenously administered TGF-beta 1 on the expression of surfactant proteins (SPs) and lipids, fatty acid synthetase, and ultrastructural morphology in human fetal lung cultured for 5 days with and without dexamethasone (10 nM). Expression of the type II cell-specific marker surfactant proprotein C (proSP-C), studied by [35S]Met incorporation and immunoprecipitation, increased sevenfold with dexamethasone treatment. TGF-beta 1 (0.1-100 ng/ml) in the presence of dexamethasone inhibited 21-kDa proSP-C expression in a dose-dependent manner (maximal inhibition 31% of control level at 100 ng/ml). There was no change in [35S]Met incorporation into total protein in any of the treatment groups vs. the control group. In immunoblotting experiments, TGF-beta 1 blocked culture-induced accumulation of SP-A and SP-B. Under the same conditions, TGF-beta 1 reduced mRNA content for SP-A, SP-B, and SP-C to 20, 38, and 41%, respectively, of matched control groups but did not affect levels of beta -actin mRNA. SP transcription rates after 24 h of exposure to TGF-beta 1 were reduced to a similar extent (20-50% of control level). In both control and dexamethasone-treated explants, TGF-beta 1 (10 ng/ml) also decreased fatty acid synthetase mRNA, protein, and enzyme activity and the rate of [3H]choline incorporation into phosphatidylcholine. By electron microscopy, well-differentiated type II cells lining potential air spaces were present in explants cultured with dexamethasone, whereas exposure to TGF-beta 1 with or without dexamethasone resulted in epithelial cells lacking lamellar bodies. We conclude that exogenous TGF-beta 1 disrupts culture-induced maturation of fetal lung epithelial cells and inhibits expression of surfactant components through effects on gene transcription.

transforming growth factor-beta 1; human fetal lung explants; surfactant proteins; fatty acid synthetase; dexamethasone

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

PULMONARY SURFACTANT is a surface-active mixture of phospholipid and protein secreted by the alveolar type II cell that reduces surface tension at the air-liquid interface and allows for maintenance of alveolar stability at low lung volumes (32). The predominant lipid synthesized is dipalmitoylphosphatidylcholine, which accounts for much of the surface activity. The biosynthetic pathway for dipalmitoylphosphatidylcholine is complex, but it appears to involve both de novo synthesis as well as remodeling of previously synthesized lipid components. A key enzyme involved in the synthesis of new phospholipid is fatty acid synthetase (FAS), a rate-limiting and hormonally regulated enzyme that catalyzes conversion of malonyl-CoA to fatty acid (31). In addition to phospholipids, surfactant also contains several unique proteins designated surfactant protein (SP) A, SP-B, SP-C, and SP-D (reviewed in Refs. 3, 32). SP-A regulates lipid secretion and uptake in vitro and contributes to local host defense. Two other, smaller lipophilic proteins, SP-B and SP-C, each confer properties of rapid surface adsorption to synthetic phospholipid mixtures. In humans, the expression of SP-C is limited to the alveolar type II cell, whereas SP-A and SP-B expression have been shown to be more promiscuous, having also been documented in other airway epithelia (27). SP-D, like SP-A, is also important for local lung host defense but has not been shown to play a role in surfactant homeostasis or in the modulation of its biophysical function.

A developmental deficiency of surfactant is the major cause of respiratory distress syndrome (RDS) in premature infants (1), and there is an emerging body of evidence that dysfunction of the surfactant system (either a relative deficiency or inactivation of normal biophysical properties) also contributes to the pathogenesis of both congenital and acquired lung disease (45). Inherited SP-B deficiency as well as conditions such as acute RDS and Pneumocystis carinii pneumonia is associated with alterations in surfactant lipids, proteins, and biophysical function (10, 14, 17, 45).

RDS can progress to chronic lung disease (CLD). Although many factors contribute to the development of CLD, pulmonary inflammation may have a central role in the pathogenesis (2, 15, 19, 20, 47). Recently, an association between an early postnatal rise in bronchoalveolar lavage levels of the multifunctional cytokine transforming growth factor (TGF)-beta 1 and the development of CLD has been reported (20). TGF-beta 1 has also been associated with the increased expression of extracellular matrix genes observed in biopsy specimens from adult humans with idiopathic pulmonary fibrosis (6) and implicated as a mediator of the type II cell proliferation seen in the bleomycin-treated rat model of lung injury and repair (18).

TGF-beta 1 is a cytokine notable for its capacity to modulate a variety of cellular behaviors (reviewed in Ref. 13). It is a member of a superfamily of cytokines that includes bone morphogenetic proteins, mullerian inhibitory substance, and activin, all of which regulate cellular differentiation and matrix deposition in tissue repair. The three isoforms of TGF-beta (beta 1, beta 2, and beta 3) are structurally related, with a high level of sequence homology and nearly identical biological activities. Analysis of cDNAs has demonstrated that each is initially synthesized as a larger precursor molecule containing the mature form of TGF-beta at the carboxy-terminal region, which is then proteolytically cleaved, secreted as an inactive homodimer (latent TGF-beta ), and then activated extracellularly before receptor signaling.

Studies (16, 33) using fetal mouse lung have shown that TGF-beta 1 may play a central regulatory role in lung morphogenesis and differentiation. TGF-beta 1 is expressed as early as day 11 of gestation in murine lung and colocalizes with various extracellular matrix proteins including collagen types I and II expressed at the epithelial-mesenchymal interfaces of stalks and clefts of the developing lung (16). Immmunoreactive TGF-beta 1 increases between days 14 and 15 during differentiation of primordial tubules into alveolar and bronchiolar ducts. Exogenous TGF-beta 1 has been shown to inhibit branching morphogenesis in cultured murine embryonic lung in a concentration-dependent manner (33). Furthermore, both epithelial cell differentiation and lung sacculation are delayed in transgenic mice bearing a chimeric gene composed of human SP-C gene promoter and porcine TGF-beta 1 cDNA (49), whereas targeted disruption of the TGF-beta 1 gene is associated with development of inflammatory infiltrates in the postnatal lung of homozygous knockout mice (36). These data suggest that although TGF-beta 1 may be involved in normal growth and development of lung epithelial cells, changes in spatial distribution and/or absolute levels of expression can affect epithelial cell differentiation and organogenesis (37).

Overexpression of TGF-beta 1 has been shown to have additional effects on epithelial cell differentiation and surfactant component expression. The arrested morphogenesis observed in the lungs from transgenic mice bearing the SP-C-TGF-beta 1 gene was accompanied by decreased immunostaining of the epithelial cells for surfactant proprotein (proSP) C and Clara cell secretory proteins (49). Exogenous TGF-beta 1 added to alveolar type II cells isolated from the adult rat further accelerated the culture-induced loss of SP-C mRNA (24). Inhibition of SP-A expression has been observed in a human bronchiolar adenocarcinoma cell line treated with exogenous TGF-beta , but the mechanism for the alteration of mRNA content is undefined.

The human fetal lung explant model has been used to study the expression and processing of SP-A, -B, and -C in early gestation (3, 4, 23, 34, 35, 40, 42-44). Although various factors have been shown to influence expression of these SPs in this model, the effect of TGF-beta 1 has not been investigated in detail. Previously, inhibition of epidermal growth factor-induced increases in SP-A in explants by a human platelet-derived preparation of TGF-beta containing all three isoforms has been published (44).

In this study, we investigated the effects of exogenously administered TGF-beta 1 on the expression of surfactant components and epithelial cell ultrastructure in a cultured human fetal lung model. Our findings indicate that TGF-beta 1 differentially downregulates the expression of three SPs acting at the level of gene transcription, inhibits the expression of a key regulatory enzyme for phospholipid synthesis, and alters epithelial cell morphology. A preliminary report of this work has been published (34).

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Materials

Recombinant TGF-beta 1 was obtained from Genentech (San Francisco, CA) and Collaborative Biomedical Products (Bedford, MA), and dexamethasone was from Sigma (St. Louis, MO). [32P]dCTP, [32P]UTP, and [methyl-3H]choline (60 Ci/mmol) were purchased from NEN (Boston, MA). Trans 35S label was obtained from ICN Flow (Costa Mesa, CA). Polyclonal SP-A, SP-B, and proSP-C antisera produced in rabbits have been previously described (4, 35, 39). Protein A-agarose was obtained from Life Technologies (Bethesda, MD). All culture medium was produced by the Cell Center Facility at the University of Pennsylvania (Philadelphia).

All other reagents were electrophoretic grade and were purchased from Bio-Rad Laboratories (Hercules, CA) and/or Sigma.

Fetal Lung Explant Culture

Human fetal lung was obtained from second-trimester therapeutic abortions under protocols approved by the Committee in Human Research at Children's Hospital of Philadelphia and the Committee on Studies Involving Human Beings at the University of Pennsylvania. Distal lung parenchyma from fetuses 19-23 wk of gestation, previously screened for the presence of maternal infection, was chopped into 1-mm pieces and cultured as explants in serum-free Waymouth medium with and without dexamethasone (10 nM). TGF-beta 1 in concentrations of 0.1, 1, 10, 30, and 100 ng/ml was separately added to each dish on day 1 in the presence and/or absence of dexamethasone. Explants were maintained at 37°C in 95% air-5% CO2 on a rocker platform as previously described (3, 4, 11, 23, 35). The medium was changed daily, and explant tissue was harvested after 5 days in culture. For studies on transcription rates, TGF-beta 1 was added to cultured explants on day 4, and nuclei were harvested on day 5 (24 h).

Metabolic Labeling

Explants were starved in Met-Cys-free DMEM for 90 min, and metabolic labeling was done by the addition of trans 35S label-DMEM reaction mixture (100-200 µmCi/ml as methionine) for up to 4 h as previously described (35). After continuous labeling, samples were harvested by removal from the labeling medium and extensive rinsing in PBS containing protease inhibitors (5 µg/ml each of leupeptin, aprotinin, and pepstatin A). Harvested samples were frozen at -70°C for further analysis.

mRNA Isolation

Total mRNA was isolated from harvested lung tissue with the TRIzol Reagent (GIBCO BRL, Gaithersburg, MD) according to the manufacturer's instructions. Briefly, tissue samples were homogenized in 1 ml of TRIzol Reagent and incubated for 5 min at 30°C. Phase separation was achieved by centrifuging samples at 12,000 g at 4°C for 15 min after the addition of 0.2 ml of chloroform. RNA was precipitated by further incubation and centrifugation after the addition of 0.5 ml of isopropanol. RNA pellets were subsequently washed with 75% ethanol and stored at -70°C.

Analytic Methods

Western blotting. For SP-A, SP-B, and FAS analysis, lung explants harvested after 5 days in culture were sonicated and homogenized, then subjected to SDS-PAGE, and the separated proteins were transferred electrophoretically overnight as previously described (4, 35). After transfer, immunoblotting was performed separately with either primary anti-SP-A (1:5,000) or anti-SP-B (1:5,000) antisera and secondary goat anti-rabbit IgG antisera conjugated with horseradish peroxidase (1:10,000). Similarly, FAS protein was detected with a polyclonal anti-rat FAS antiserum (1:3,000) after explant proteins were separated on 5% SDS-PAGE. In each case, immunoreactivity was detected by enhanced chemiluminescence (ECL) with the ECL Detection Kit (Dupont-NEN, Bedford, MA) according to the manufacturer's instructions.

Determination of protein content. Total protein content of lung tissue samples was determined by the Bradford (5) method with bovine IgG as the standard.

Quantitative immunoblot assay. Tissue levels of SP-A and SP-B were measured with a quantitative immunoblot assay as previously published (4). Briefly, fetal lung tissue was sonicated in a solution of protease inhibitors containing 10 mM benzamidine, 50 mM N-ethylmaleimide, and 10 mM phenylmethylsulfonyl fluoride. Samples of sonicated tissue (200-µl total volume) of known protein content were serially diluted in PBS, pH 7.5, and spotted onto nitrocellulose paper with a vacuum-assisted 96-well dot-blot apparatus (Bio-Rad). After the blots were dried and blocked with 2% milk, they were subsequently incubated with either primary anti-SP-A or anti-SP-B followed by a secondary goat anti-rabbit IgG-conjugated with horseradish peroxidase. Blots were washed extensively and exposed by ECL. The net density in each spot was determined by densitometric scanning with film exposures within the linear range. Results were normalized to SP-A or SP-B in a single pooled human surfactant prepared in our laboratory that was used as an internal standard. Assays were performed in duplicate on tissue from duplicate culture dishes, and protein content was read from the linear portion of the standard and unknown sample curves.

Immunoprecipitation. For proSP-C analysis, radiolabeled fetal lung homogenates were solubilized in buffer containing nonionic detergent and protease inhibitors and immunoprecipitated with human proSP-C antiserum as previously described (35). Proteins liberated by heating at 100°C for 15 min were separated by one-dimensional 16.5% SDS-PAGE with a Tris-tricine buffer system. Separated proteins were electrophoretically transferred to 0.2-mm nitrocellulose at 60 mA/cm2 for 12-18 h and visualized by autoradiography. 35S counts in specific immunoprecipitated bands were quantitated by direct beta -scanning of the blots with an AMBIS 4000 radioanalyzer (Scanolytics, San Diego, CA).

FAS activity. The activity of FAS (EC 2.3.1.85) was assayed by the incorporation of radioactive malonyl-CoA with the method of Roncari (30) as modified and described (12). Explants were sonicated in cold water and centrifuged at 13,000 rpm for 15 min, and aliquots of the supernatants were assayed by a 15-min incubation in the presence of 50 µM malonyl-CoA and 30 µM acetyl-CoA with 0.005 µCi [14C]malonyl-CoA in a 0.5-ml reaction volume. Enzyme activity, initiated by the addition of 50 µM NADPH, is expressed as nanomoles of malonyl-CoA utilized per minute per milligram of protein. All samples were assayed in duplicate.

Choline incorporation into phosphatidylcholine. To quantitate the rate of choline incorporation into phosphatidylcholine (PC), explants were cultured for the last 4 h in the presence of [3H]choline (1 µCi/ml medium), and then total lipid was extracted and PC was isolated by thin-layer chromatography on Whatman LK5D plates with chloroform-methanol-NH4OH (7 N; 60:35:5 vol/vol/vol) as previously described (11).

Quantitation of mRNA levels. Specific mRNA content was determined by cDNA hybridization with Northern and dot-blot analyses as previously described (28). Full-length human cDNA inserts containing SP-A, SP-B, SP-C, and beta -actin have been previously used as probes by our group (23, 28). A human cDNA probe (clone Pg8) containing a 2-kb fragment complementary to the 3'-end of the FAS coding region (7) was a generous gift from Dr. D. Chalbos (Faculty of Medicine, INSERM, Montpellier, France). [32P]cDNAs for SP-A, SP-B, SP-C, FAS, and beta -actin were prepared from purified plasmid inserts by labeling with [32P]dCTP with a random-primer labeling technique (Ready-To-Go Kit, Pharmacia, Piscataway, NJ). Nitrocellulose blots were hybridized under high stringency as previously published (23, 28) and then exposed to Kodak XAR for 1-7 days at -70°C. Counts in specific mRNA bands and dots were quantitated either by utilizing direct beta -counting with an AMBIS 4000 radioanalyzer (Scanolytics) or by densitometric scanning of exposed film and quantitation with Quantity 1 (PDI, Huntington Station, NY).

Nuclear transcription elongation (run-on) assay. The methods for the assay have been previously described (28). Nuclei were isolated from explants by homogenization and centrifugation. Nuclei (2.5 × 107) were resuspended in reaction buffer and incubated at 37°C for 30 min with 0.25 mM GTP, 0.25 mM CTP, 0.5 mM ATP, 100 µCi of [32P]UTP (3,000 Ci/mmol), and 40 U of RNase inhibitor. Digestion of the nuclei was done by successively adding RNase-free DNase I and proteinase K. RNA was prepared, and free nucleotides were removed with gel-filtration (Sephadex G-50) spin columns.

After linearization and denaturation, unlabeled human cDNAs for SP-A, SP-B, SP-C, and beta -actin in Bluescript plasmid were applied to a nitrocellulose filter strip (5 µg/dot) with a dot-blot apparatus. The filters were hybridized with 2.5 × 106 counts/min of RNA at 42°C for 4 days, washed under high-stringency conditions, and incubated with RNase. The filters were exposed to X-ray film for 2-7 days at -70°C, and autoradiograms were quantified by densitometric scanning.

Electron microscopy. Fetal lung explants were fixed in 2.5% glutaraldehyde-0.1 M sodium cacodylate (pH 7.2) for 3 h at 4°C, then rinsed in 0.1 M sodium cacodylate buffer (pH 7.2), and postfixed in 1% osmium tetroxide (12). After dehydration through a series of ethanols and propylene oxide, the tissues were embedded in epoxy resin. Ultrathin sections were cut with a diamond knife, contrasted with uranyl acetate and lead citrate, and evaluated in a JEOL CX100II transmission electron microscope operated at 80 kV.

TCA precipitation. Radiolabeled fetal lung tissue harvested at specified time points was sonicated and centrifuged at 14,000 g for 10 min. Aliquots of the supernatant were precipitated in 10% TCA, and the amount of label incorporated into acid-precipitable proteins was analyzed as previously described (35).

Statistics

Statistical analyses for group mean data were carried out with SigmaStat (Jandel Scientific). Comparisons of multiple groups by ANOVA were made with either Fisher's protected least significant difference or Student-Newman-Keuls test of normality. The level of significance was P < 0.05.

    RESULTS
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Procedures
Results
Discussion
References

Content of SPs

The treatment of cultured human fetal lung with TGF-beta 1 modulated the expression of all three major SPs. In Fig. 1, TGF-beta 1 inhibition of tissue SP-A content, previously shown to increase many times during explant culture, was demonstrated by two different immunoblotting methods. By Western blot analysis, TGF-beta 1 blocked the culture-induced expression of SP-A comparable to the previously described inhibitory effect with dexamethasone (Fig. 1A) (23, 42). This response was quantitated by immunodot blotting that demonstrated that TGF-beta 1 inhibition of total immunoreactive SP-A was dose dependent (Fig. 1B).


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Fig. 1.   Transforming growth factor (TGF)-beta 1 inhibits expression of surfactant protein (SP) A in human fetal lung. A: representative Western blot analysis of SP-A expression in lung homogenate samples harvested from cultured human fetal lung. Second-trimester fetal lung cultured in presence and absence of dexamethasone (Dex) was treated with TGF-beta 1 for 4 days. Sample pairs for each condition represent separate protein samples from duplicate dishes from the same lung cultures. Each lane contains 50 µg of total protein. Expression of SP-A was detected with polyclonal SP-A antisera with an enhanced chemiluminescence system (ECL) as described in EXPERIMENTAL PROCEDURES. Shown is a 26- to 36-kDa SP-A band (SP-A26-36). Both Dex and TGF-beta 1 decreased SP-A content. B: quantitative dot-blot analysis. Lung homogenates from 3 separate experiments were analyzed with solid-phase immunodot-blot analysis as described in EXPERIMENTAL PROCEDURES. Data normalized as percentage of 5-day control culture are expressed as means ± SE. * P < 0.05 vs. control culture. + P < 0.05 vs. Dex.

TGF-beta 1 also inhibited the expression of SP-B in cultured lung. Through a combination of transcriptional and posttranslational mechanisms, culture of human fetal lung induces both de novo synthesis and processing of 42-kDa proSP-B (proSP-B42) to mature 8-kDa SP-B (SP-B8), and these effects are markedly enhanced by inclusion of dexamethasone in the culture medium (4). Under both control and dexamethasone conditions, TGF-beta 1 (100 ng/ml) completely blocked the expression of SP-B8 (Fig. 2A). Quantitative immunodot blotting demonstrated dose dependence of the TGF-beta 1 effect on total SP-B immunoreactivity, with significant inhibition at 10 ng/ml (Fig. 2B).


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Fig. 2.   TGF-beta 1 inhibits expression of SP-B in human fetal lung. A: representative Western blot analysis of SP-B expression in lung homogenate samples harvested from cultured human fetal lung. Second-trimester fetal lung cultured in presence and absence of Dex was treated with TGF-beta 1 for 4 days. Sample pairs for each condition represent separate protein samples from duplicate dishes from the same lung cultures. Each lane contains 50 µg of total protein. Expression of SP-B was detected with polyclonal SP-B antisera with ECL system as described in EXPERIMENTAL PROCEDURES. Shown is an 8-kDa SP-B band (SP-B8), which constituted majority of immunoreactive protein identified on blot. B: quantitative dot-blot analysis. Data from 3 experiments were normalized as percentage of 5-day control culture and are expressed as means ± SE. * P < 0.05 vs. control culture. + P < 0.05 vs. Dex.

SP-C is a type II cell-specific protein considered to be a specific marker of type II epithelial cell differentiation (27). Analysis of mature SP-C expression has been hampered by its poor antigenicity, but Solarin et al. (35) previously reported the usefulness of proSP-C21 as a surrogate marker of SP-C expression. Human fetal lung explants cultured for 5 days in the presence and absence of dexamethasone were metabolically labeled with [35S]Met-Cys and immunoprecipitated with a proSP-C antiserum to identify synthesis of the 21-kDa SP-C primary translation product. Dexamethasone induced a marked upregulation of proSP-C21 expression compared with control cultures (Fig. 3A). The addition of TGF-beta 1 with dexamethasone on day 1 inhibited dexamethasone-stimulated synthesis of proSP-C21. Quantitation of 35S in the immunoprecipitated bands demonstrated that dexamethasone alone increased proSP-C expression almost sevenfold compared with the control bands (Fig. 3B). Inhibition by TGF-beta 1 was dose dependent, with a half-maximal response at ~10-20 ng/ml and complete inhibition occurring between 30 and 100 ng/ml. The magnitude of the TGF-beta 1 effect on proSP-C expression increased with exposure time (Table 1).


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Fig. 3.   TGF-beta 1 inhibits synthesis of 21-kDa surfactant proprotein C (proSP-C21) in human fetal lung. A: human fetal lung cultured for 4 days in presence and absence of 10 nM Dex and increasing concentrations of TGF-beta 1 (in ng/ml) as indicated was metabolically labeled with [35S]Met-Cys as described in EXPERIMENTAL PROCEDURES. One hundred micrograms of each lung homogenate were immunoprecipitated with anti-NPROSP-C antisera. Captured proteins were analyzed by SDS-PAGE and transferred to nitrocellulose. This representative autoradiogram from 1 experiment shows a dose-dependent decrease in proSP-C21 expression induced by TGF-beta 1. B: data from several experiments were analyzed for 35S incorporation into proSP-C21. Each band identified was quantitated by direct beta -counting with an Ambis 4000 radioanalyzer. Nos. in parentheses, concentration of TGF-beta 1 (in ng/ml) in medium; nos. in bars; no. of experiments/condition. Data were normalized as percentage of 5-day control culture and are expressed as means ± SE for conditions containing multiple experiments and as arithmetic mean for conditions containing 2 experiments: Dex + 1 ng/ml TGF-beta 1 = 378-645% of 5-day control value and Dex + 30 ng/ml TGF-beta 1 = 76.3-99.7% of 5-day control culture. * P < 0.05 vs. control culture. + P < 0.05 vs. Dex alone.

                              
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Table 1.   Time-dependent effect of TGF-beta 1 on proSP-C21 synthesis

The observed effects of TGF-beta 1 on SP expression were not a consequence of a global inhibition of total protein synthesis by cytokine treatment. After 4 h of continuous labeling, incorporation of [35S]Met-Cys into total protein (measured as 35S dpm/µg total protein) for dexamethasone- and TGF-beta 1-treated explants was determined and compared with control explants. There was no significant difference in total 35S specific activity for control, dexamethasone, or dexamethasone + TGF-beta 1 tissues (control, 31,191 ± 5,910 dpm/µg, n = 5; dexamethasone, 37,058 ± 8,140 dpm/µg, n = 5; dexamethasone + 10 ng/ml of TGF-beta 1, 32,954 ± 3,107 dpm/µg, n = 3; dexamethasone + 100 ng/ml of TGF-beta 1, 42,578 ± 2,974 dpm/µg, n = 3; P > 0.05 for all groups vs. control group by ANOVA). Similarly, the percentage of TCA-precipitable 35S counts was unaffected by all of the treatment combinations (data not shown).

SP mRNA Content and Transcription Rate

Figure 4 is a representative Northern blot analysis demonstrating the effects of TGF-beta 1 on SP mRNA levels in both control and dexamethasone-treated fetal lung cultures. As for protein expression, 10 nM dexamethasone inhibited SP-A mRNA content and greatly stimulated levels of message for both SP-B and SP-C. In the absence of dexamethasone, TGF-beta 1 at 10 ng/ml substantially decreased the mRNA signal for all three SPs. Explants cultured in the presence of dexamethasone required higher doses of TGF (100 ng/ml) to achieve the same magnitude of inhibition of expression of SP-B and SP-C.


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Fig. 4.   Northern blot analysis of SP expression in human fetal lung. Total lung RNA was prepared from human fetal lung cultured for 5 days in presence and absence of Dex and/or TGF-beta 1 at doses (in ng/ml) indicated. Ten micrograms of total RNA were separated by electrophoresis, transferred to nitrocellulose membranes, and probed with 32P-labeled probes against SP-A and SP-C. Bands visualized by autoradiography represent 0.9-kb SP-C and 1.6-kb SP-A. Blots were then stripped and reprobed under identical methodology for SP-B to detect 2.0-kb SP-B transcript. Efficiency of loading was confirmed by reprobing blots with 32P-labeled beta -actin cDNA. Bands were quantitated by direct beta -scanning with an AMBIS 4000 radioanalyzer. In this representative experiment, inhibition of SP-B and SP-C by 10 ng/ml of TGF-beta 1 in presence of Dex was modest (corrected for actin SP-B = 94% of Dex alone; SP-C = 85% of Dex alone), but higher doses of TGF-beta 1 (100 ng/ml) produced substantial inhibition of both SP-B and SP-C (corrected for actin, SP-B = 29% of Dex alone and SP-C = 11% of Dex alone). Data from multiple independent experiments are summarized in Table 2.

Quantitation of the downregulation of SP mRNA content by TGF-beta 1 was performed by analysis of data from multiple independent experiments (Table 2). TGF-beta 1 at a concentration of 10 ng/ml substantially inhibited SP-A in both control and dexamethasone-treated cultures. At the concentrations used, TGF-beta 1 alone was more inhibitory than dexamethasone for SP-A mRNA (P < 0.05), and these responses were not additive. TGF-beta 1 (10 ng/ml) also produced significant decreases in the levels of both SP-B and SP-C mRNAs in cultures containing 10 nM dexamethasone (P < 0.05 vs. dexamethasone alone). Although SP-B mRNA was also downregulated by TGF-beta 1 in the absence of dexamethasone, a significant effect on the already low basal expression of SP-C mRNA could not be detected in 5-day control cultures.

                              
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Table 2.   Effect of TGF-beta 1 on SP mRNA expression

The mechanism for TGF-beta 1 inhibition of SP and SP message was further investigated with nuclear run-on assays of gene transcription. Figure 5A shows representative results for SP-A, SP-B, and SP-C. To avoid the effects of prolonged exposure to TGF-beta 1 on cellular differentiation, explants were cultured for 4 days in the presence of dexamethasone, and then TGF-beta 1 was added during the final 24 h of culture. Under these conditions, explants exposed to TGF-beta 1 showed ~80% inhibition of the transcription rate for both SP-A and SP-C and ~50% inhibition for SP-B (Fig. 5B).


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Fig. 5.   Effect of TGF-beta 1 on transcription rate. A: representative autoradiogram showing duplicate samples of explants from a 23-wk lung cultured in medium for 4 days, then exposed to TGF-beta 1 (30 ng/ml) for 24 h. Nuclei were harvested as described in EXPERIMENTAL PROCEDURES, and rate of gene transcription for SP-A, SP-B, and SP-C as well as for beta -actin was measured by hybridization to specific cDNA probes as previously published (28). BS, negative control consisting of Bluescript plasmid alone. B: data from 6 samples from 2 lungs were normalized to results for beta -actin and are expressed as means ± SE of ratio of treated to control transcription rate.

Choline Incorporation and FAS Expression

Addition of exogenous TGF-beta 1 resulted in the disruption of normal surfactant phospholipid synthetic metabolism in cultured lung as assessed by choline incorporation into total PC, FAS protein expression, FAS enzyme activity, and FAS mRNA expression. Dexamethasone alone induced a substantial increase in [3H]choline incorporation into PC, whereas a 4-day exposure to TGF-beta 1 produced a dose-dependent inhibition of this incorporation in both control and dexamethasone-treated tissues (Table 3).

                              
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Table 3.   Effect of TGF-beta 1 on choline incorporation

FAS represents an important regulatory enzyme in the de novo synthetic pathway of surfactant PC (31). Western blots of lung homogenates from explants treated with dexamethasone and/or TGF-beta 1 were probed with a polyclonal anti-FAS antibody, and immunoreactive protein content was quantitated by densitometry. In parallel with data for choline incorporation, dexamethasone stimulated, whereas TGF-beta 1 suppressed, the level of FAS protein in lung homogenates (Table 4). Similar changes in FAS enzyme activity were also observed in both control and dexamethasone-treated explants (Table 4).

                              
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Table 4.   Effect of TGF-beta 1 on FAS expression

The changes in FAS protein content and enzyme activity were further reflected in similar reductions in FAS mRNA content. By Northern blot analysis, downregulation of the 9.0-kb FAS mRNA band was observed in both control and dexamethasone-treated explants after the administration of 10 ng/ml of TGF-beta 1, with a higher dose (100 ng/ml) completely abolishing the FAS mRNA signal (Fig. 6). Quantitation of the modulation of FAS mRNA expression by TGF-beta 1 treatment was performed by dot-blot analysis of RNA samples from multiple independent experiments and indicated an equivalent percent decrease in both control and dexamethasone-treated explants exposed to 10 ng/ml of TGF-beta 1, and a greater response to 100 ng/ml of TGF-beta 1 in the presence of dexamethasone (Table 4).


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Fig. 6.   Effect of TGF-beta 1 on fatty acid synthetase (FAS) mRNA expression. Representative Northern blot analysis shows FAS expression in explants cultured for 5 days in absence of hormones (control) and in presence of 10 nM Dex alone or in combination with TGF-beta 1 (10 or 100 ng/ml). Total RNA was isolated and probed for FAS and beta -actin content with 32P-labeled cDNA probes as detailed in EXPERIMENTAL PROCEDURES. Each lane contains 10 µg of total RNA. Quantitation of FAS mRNA expression from multiple independent experiments performed by dot-blot hybridization (described in EXPERIMENTAL PROCEDURES) is summarized in Table 4.

Exogenous TGF-beta 1 Alters Explant Ultrastructural Morphology

Based on the results presented in Figs. 1-6 and Tables 1-4 demonstrating that TGF-beta 1 induced changes in SP and lipid synthesis, we hypothesized that TGF-beta 1 blocked ultrastructural changes of pulmonary epithelial cell differentiation. To examine this question, ultrathin sections of cultured human fetal lung were prepared for electron-microscopic analysis. Second-trimester lung tissue sectioned before culture demonstrated a pattern of potential air spaces lined by homogeneous columnar epithelial cells devoid of distinct type II cell morphological characteristics (Fig. 7A). As previously reported (11), culture of the lung for 5 days initiated a pattern of increased differentiation characterized by increases in air space size, appearance of multilamellated bodies in epithelial cells, and a decrease in cellular glycogen stores (Fig. 7B). This process of culture-induced cytodifferentiation was further enhanced by the addition of 10 nM dexamethasone that increased both the number and size of lamellar bodies and the size of microvilli on the apical plasma membrane (Fig. 7C).


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Fig. 7.   TGF-beta 1 blocks ultrastructural maturation of cultured human fetal lung. Ultrathin sections from second-trimester human fetal lung were prepared and stained as described in EXPERIMENTAL PROCEDURES. A: preculture. B: 5-day control culture. C: 5-day culture with 10 nM Dex. D: 5-day culture with 10 ng/ml of TGF-beta 1. E: 5-day culture with 10 nM Dex + 10 ng/ml of TGF-beta 1. F: 5-day culture with Dex + 100 ng/ml of TGF-beta 1. Lamellar bodies (arrows) were induced by explant culture and further enhanced by inclusion of dexamethasone. L, lumen of potential air space. Presence of apoptotic bodies, vacuolization of epithelial cells, and occasional ciliated epithelial cells were also noted in TGF-beta 1-treated explants (data not shown).

The commensurate administration of TGF-beta 1 at doses shown to block SP and SP mRNA expression (10-100 ng/ml) markedly inhibited the stimulatory effect of culture (Fig. 7D) and dexamethasone (Fig. 7, E and F) on lung maturation. Samples treated with TGF-beta 1 showed a striking disruption of normal differentiated type II epithelial cell characteristics, including a loss of lamellar bodies and retention of cellular glycogen.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

TGF-beta 1 has previously been shown to modulate normal lung development and is implicated as a mediator in the pathogenesis of CLD of the newborn (16, 20, 33, 49). In a series of experiments designed to test the effect of TGF-beta 1 on human lung epithelial cell function and differentiation, we administered recombinant human TGF-beta 1 to fetal lung explants and evaluated the effects on cell-specific biochemical and morphological markers of the type II cell phenotype. The results presented here clearly demonstrate that TGF-beta 1 has profound effects on the expression of both protein and lipid components of the surfactant system. In addition, ultrastructural studies confirm a marked effect of TGF-beta 1 on epithelial cell morphology.

In our experimental model, exogenous TGF-beta 1 was delivered directly to cultured lung explants, a stable, well-characterized model for testing the effects of hormones, cytokines, and other agents on SP and lipid metabolism (3, 4, 11, 12, 23, 34, 35, 40, 42-44). In the absence of hormone and serum, the process of culturing lung explants for 5 days leads to thinning of the mesenchyme, expansion of potential air spaces, and differentiation of epithelial cells as characterized by the development of microvilli and primordial lamellar bodies and by an increased content of surfactant lipid and proteins (11). Our group and others have found that the content of SP-A and SP-B increases (4, 23, 40, 43), whereas SP-C mRNA and proSP-C remain relatively low throughout the 5-day culture period, even when late second-trimester explants are utilized (23, 35, 43). For both SP-B and SP-C, dexamethasone treatment increases mRNA content, protein synthesis, and posttranslational processing of the resulting proproteins (4, 23, 35, 40, 43). This enhanced expression of SP-B and SP-C is confined exclusively to the air space epithelia that eventually undergo morphological differentiation to type II cells. In contrast, SP-A, normally upregulated by culture alone, is suppressed by continued exposure to dexamethasone, with a biphasic dose-response curve (3, 42). Each of these patterns of SP expression was reiterated by the current results reported here for explants cultured in the absence of TGF-beta 1. Although a potential limitation of this model is an inability to target specific cellular subpopulations within the explant, similar responses to glucocorticoids have been observed in vivo and support the physiological relevance of the human fetal lung culture system as a model to test the effects of cytokines.

The effect of TGF-beta 1 on SP-A expression has been examined previously with cultured H441 cells (41). At a dose of 1 ng/ml, TGF-beta 1 completely inhibited the expression of SP-A in this Clara cell-like cell line. Our results showing inhibition of SP-A expression by TGF-beta 1 in the explant system confirm this finding and provide additional evidence for the physiological relevance of this cytokine in the modulation of SP expression. The higher doses required for maximal inhibition of SP-A expression in the present studies are likely due to an increased diffusion distance in the explants and/or the presence of receptors on non-type II cells competing for TGF-beta 1. Additionally, as yet undefined differences in signal transduction events or TGF-receptor physiology between a transformed cell line and human fetal lung may play a role.

In the present work, we demonstrated that TGF-beta 1 could also inhibit expression of the two hydrophobic SPs. In both the presence and absence of glucocorticoid, TGF-beta 1 inhibited the expression of the mature SP-B8. Because of the low basal levels of proSP-C routinely observed in explant cultures lacking exogenous steroids (35), we chose to study the effects of TGF-beta 1 on SP-C under culture conditions containing dexamethasone. TGF-beta 1 (at concentrations of 10-100 ng/ml) was inhibitory even in the presence of stimulating doses of dexamethasone. Our results in the cultured human fetal lung confirm previous findings for SP-C in vivo. Zhou et al. (49) observed that lung-specific overexpression of TGF-beta 1 inhibited immunohistochemical staining for proSP-C in lungs of mice containing a TGF-beta 1 transgene. In an in vitro system (primary adult rat type II cell cultures), Maniscalco et al. (24) used in situ hybridization to demonstrate an inhibitory effect of exogenous TGF-beta 1 on SP-C mRNA levels. Because of an inherent phenotypic instability of isolated type II cells, in which the levels of SP-C mRNA typically decline with time in culture on plastic, interpretation of the effects of TGF-beta 1 on mRNA in that system should be made with caution.

The quantitative effects of TGF-beta 1 on SP mRNA content paralleled the decreases observed in the levels of protein and were accompanied by similar inhibitions of the rates of gene transcription for each SP (Fig. 5). The underlying molecular events of TGF-beta 1-induced inhibition of SP gene transcription are not fully characterized. Because the cultured human fetal lung is a complex multicellular model, alterations of transcription rates after TGF-beta 1 administration could involve indirect effects potentially mediated either by changes in cellular composition or through changes induced in other cellular constituents (e.g., fibroblasts). However, based on several lines of evidence, it is more likely that the effects of TGF-beta 1 are a result of its direct interaction with the epithelial cell. First, Torday and Kourembanas (38) previously demonstrated that a TGF-beta homologue secreted by fetal lung fibroblasts can inhibit phospholipid synthesis in cultured fetal type II cells. Second, TGF-beta 1 has been shown to decrease SP-A and SP-B gene expression in H441 cells (41). Third, in preliminary studies using plasmid transfections, we have found that TGF-beta 1 (10 ng/ml), acting through a protein kinase C (calphostin C-inhibitable) pathway, downregulates SP-B promoter activity in H441 cells (21). The effects of TGF-beta 1 on SP gene and SP expression are reminiscent of those observed with 12-O-tetradecanoylphorbol 13-acetate in both explants and H441 cells (28, 29). We have found that both 12-O-tetradecanoylphorbol 13-acetate and TGF-beta 1 cause cytoplasmic trapping and loss of both thyroid transcription factor-1 and hepatocyte nuclear factor-3 from the nucleus of treated H441 cells (21, 22). It has previously been shown that these transcription factors are important in SP gene expression and epithelial cell differentiation (9, 46). Studies designed to address the specific postreceptor pathway mediating these changes are currently under further investigation.

We report the new observation that TGF-beta 1 downregulates the expression of FAS, an important regulatory enzyme in the synthetic pathway for the production of surfactant phospholipids (31, 37). Similarly, choline incorporation into surfactant PC was markedly decreased by TGF-beta 1. This finding is consistent with a previously published study (38) using conditioned medium containing TGF homologues from rat fetal lung fibroblasts to inhibit lipid synthesis by cultured fetal rat type II cells. The decrease in lipid synthesis mediated by TGF-beta 1 in this model likely reflects, at least in part, a functional consequence of the decrease in FAS enzyme activity. The mechanism by which TGF-beta 1 affects FAS is presently unknown but could reflect either a direct action on FAS gene expression or an effect secondary to TGF-beta 1-induced changes in other genes and/or epithelial cell differentiation.

The effects of TGF-beta 1 on lung epithelial differentiation and function have not been widely studied. The overexpression of TGF-beta 1 in lung epithelial cells of transgenic mice with the human SP-C promoter induced a neonatal lethal phenotype, with delayed structural development and inhibition of expression of two epithelial cell markers, Clara cell secretory protein and SP-C (49). In our culture system, exogenous TGF-beta 1 inhibited the normal ultrastructural maturation of the lung epithelium induced by culture and by dexamethasone. Electron microscopy (Fig. 7) demonstrated that lung explants cultured with TGF-beta 1 failed to develop the normal number or appearance of lamellar bodies, a well-characterized phenotypic marker of type II cells. In contrast to a lung bud system, tissue used in our study is from midgestation, when branching morphogenesis is complete and only cell differentiation is occurring (11). We observed that the majority of the cells lining the lumens of potential air spaces from the TGF-beta 1-treated explants contained substantially lower numbers of lamellar bodies in contrast to tissue cultured with or without dexamethasone. It is unlikely that the phenotypic changes in the ultrastructural morphology observed in the presence of exogenous TGF-beta 1 in this system are due to either a distortion of developmental sequences during in vitro culture of the explants or a bias in the acquisition of sections.

The lack of phenotypically normal type II cells could be secondary to the deficiency of SP-B induced by TGF-beta 1. In the syndrome of congenital SP-B deficiency (10) as well as in the SP-B knockout mouse (8), the phenotype is characterized by a lack of functional SP-B8 and associated abnormalities in lipid metabolism, absence of lamellar bodies, disordered secretion, and a block in posttranslational processing of SP-C. With the exception of an increase in proSP-C processing intermediates, which would not be expected given the downregulation of SP-C gene expression, many of these same findings have been recapitulated in the TGF-beta 1-treated lungs.

Alternatively, because TGF-beta 1 is a multifunctional cytokine, disruption of epithelial cell maturation and/or differentiation could occur apart from the inhibition of surfactant component expression. Since its discovery, TGF-beta has been shown to function as an autocrine/paracrine growth factor that exerts a variety of effects on cellular functions, including regulation of cell proliferation, differentiation, and extracellular matrix production (13). The cellular response to TGF-beta is dependent on cell type and culture conditions. For cells of epithelial origin, TGF-beta has been shown to generally act as an inhibitor of growth. The proliferative response of alveolar type II cells has been shown to be inhibited by TGF-beta 1 during bleomycin-induced lung injury in rats (18). The effect of TGF-beta on differentiation is also variable; in some cases, the cytokine acts as a suppresser of differentiation, whereas it stimulates the expression of a differentiated phenotype in others. In the lung, detailed studies of these effects are limited, but the administration of TGF-beta has been shown in vitro to induce squamous metaplasia (differentiation) in human bronchial epithelial cells (26). Thus the morphological and biochemical effects seen with TGF-beta 1 administration could result from inhibition of the normal progression of differentiation to type II cells during culture or the promotion of differentiation toward another cell phenotype (e.g., type I cell). The mechanism(s) by which TGF-beta 1 affects growth and differentiation is presently not defined. TGF-beta type II receptors are present in the epithelial lining of the developing airway (48). The biological effects occurring downstream from the receptor-ligand complex may involve several possible signal transduction mechanisms, including regulation of N-myc expression, modulation of protein kinase activity, receptor-mediated tyrosine phosphorylation, and Smad signaling (25).

Deficiency and/or dysfunction of the surfactant system has been implicated in the pathogenesis of a variety of infectious, inflammatory, and toxin-mediated lung diseases (45). The findings that TGF-beta 1 inhibits surfactant components and type II cell ultrastructural differentiation support the concept that this multifunctional cytokine contributes to the pathophysiology of acquired lung disease in part through its inhibitory effects on the lung epithelium and the surfactant system. The doses of TGF-beta 1 used in this study (10-100 ng/ml) are comparable to levels of the cytokine that have been recovered early in the course (day 7) from the lungs of RDS patients who ultimately developed CLD (20). Our data support further examination of the possible role of surfactant deficiency in the pathogenesis of acquired lung disease.

    ACKNOWLEDGEMENTS

We thank Scott Russo, John Gonzales, Sree Kumar, and Yue Ning for technical assistance. We thank Sylvia Decker for performing electron microscopy.

    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute (NHLBI) Grant HL-02869 (to M. F. Beers); NHLBI Specialized Center of Research Grant 1-P50-HL-56401 (to P. Ballard, M. F. Beers, S. H. Guttentag, L. W. Gonzales, and J. Rosenbloom); National Institute of Child Health and Human Development Grant 5-P30-HD-28815 (to S. H. Guttentag); and Research Grants from the Pennsylvania Thoracic Society (to S. H. Guttentag) and the Philadelphia-Montgomery County American Lung Association (to M. F. Beers).

M. F. Beers is the recipient of a Clinician-Scientist Award from the American Heart Association.

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: M. F. Beers, Univ. of Pennsylvania School of Medicine, 524 Johnson Pavilion, 36th and Hamilton Walk, Philadelphia, PA 19104-6068.

Received 21 January 1998; accepted in final form 30 July 1998.

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