©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Cloning and Expression of Glucocorticoid-induced Genes in Fetal Rat Lung Fibroblasts
TRANSFORMING GROWTH FACTOR-beta(3)(*)

(Received for publication, July 29, 1994; and in revised form, September 22, 1994)

Jinxia Wang Maciej Kuliszewski Wendy Yee Larissa Sedlackova Jing Xu Irene Tseu Martin Post (§)

From the Medical Research Council Group in Lung Development and the Neonatal Research Division, Department of Paediatrics, Hospital for Sick Children Research Institute, University of Toronto, Toronto, Ontario M5G 1X8, Canada

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Glucocorticoids have been shown to accelerate fetal lung type II cell maturation, and this effect appears, in part, to be mediated via fibroblasts. To identify glucocorticoid induced genes in fetal lung fibroblasts, we screened a cDNA library from cortisol-treated fetal lung fibroblasts with a subtracted cDNA probe which was enriched for sequences specific for cortisol-treated fetal lung fibroblasts. Fifty-seven clones were isolated from the cDNA library. One cDNA represented approx30% of the 57 clones. Analysis of DNA sequence homology suggested that this cDNA encodes the rat transforming growth factor-beta(3) (TGFbeta(3)). We found that TGFbeta(3) mRNA was expressed in fetal lung fibroblasts but not epithelial cells. Expression of message in fetal lung fibroblasts was developmentally regulated. TGFbeta(3) mRNA levels were low during the pseudoglandular stage (day 18), peaked during the early canalicular stage of lung development (day 19), then fell again at days 20 and 21 (term = 22 days). Exposure of fetal lung fibroblasts to cortisol increased TGFbeta(3) mRNA expression in a time- and dose-dependent manner. Maternal administration of dexamethasone also enhanced mRNA expression of TGFbeta(3) in fetal lung fibroblasts. These data suggest that glucocorticoids may mediate their stimulatory effect on lung maturation by inducing TGFbeta(3) expression in fetal lung fibroblasts.


INTRODUCTION

Mesenchymal-epithelial interactions play an important role in lung morphogenesis. During the embryonic period, the mesenchyme directs and controls lung epithelial budding and branching(1) . Less is known about the influence of lung mesenchyme on alveolar epithelial differentiation during late fetal life. The pulmonary surfactant system (lipids and proteins) has been widely used as a marker for the assessment of alveolar epithelial differentiation. Significant quantities of surfactant are not synthesized until close to term. It appears that the production of surfactant is under multihormonal control, yet local cell and tissue interactions continue to modulate the endocrine signals(2) . A central role is played by endogenous fetal glucocorticoids. Glucocorticoids have been shown to accelerate the formation of pulmonary surfactant lipids and proteins, and, by inference, alveolar epithelial differentiation(1, 2, 3) . Studies using isolated fetal lung cells have demonstrated that the production of surfactant by epithelial cells is augmented by differentiation factor(s) elaborated by fetal lung fibroblasts in response to glucocorticoids(2, 3, 4) . Although the exact nature of the factor(s) remains to be elucidated, the action of glucocorticoids to induce differentiation factor(s) in fetal lung fibroblasts requires both RNA and protein synthesis(5, 6, 7) . To understand the molecular mechanism by which fibroblasts mediate the glucocorticoid action on epithelial cells during late fetal lung development, we sought to determine the glucocorticoid-inducible genes in fetal rat lung fibroblasts. Using subtractive hybridization, we cloned several cDNAs representing genes induced by glucocorticoids in fetal lung fibroblasts. In the present study, we identified TGFbeta(3)(^1)as an important developmental and glucocorticoid-inducible gene in fetal lung fibroblasts.


EXPERIMENTAL PROCEDURES

Materials

Female (200-250 g) and male (250-300 g) Wistar rats were purchased from Charles River (Quebec, Canada) and bred in our animal facilities. The sources of all cell culture materials have been described elsewhere(8) . Rat TGFbeta1 (insert size, 983 bp) and mouse TGFbeta2 (insert size, 1037 bp) cDNAs were kindly provided by Drs. S. W. Qian and F. Denhez (Laboratory of Chemoprevention, NIH/NCI, Bethesda). Human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA (insert size: 1.2 kb) was purchased from the American Type Culture Collection (Rockville, MD). Rat beta-actin cDNA (insert size 0.76 kb) was generated by reverse transcriptase-PCR cloning using rat beta-actin primers (Clontech, Palo Alto, CA). Hybond N membranes, [alpha-P]dCTP, [^3H]thymidine, and [Me-^3H]choline were from Amersham Canada (Oakville, Ontario). The poly(A) RNA purification kit, cDNA synthesis kit, and T7 sequencing kit were from Pharmacia Biotech Inc. (Baie d'Urfé, Quebec). The predigested LAMBDA ZAP®II/EcoRI cloning kit was from Stratagene (La Jolla, CA). The predigested gt 10/EcoRI cloning kit was from Promega (Fisher Scientific, Toronto, Ontario). The plasmid purification kit was from QIAGEN (Chatsworth, CA). Geneclean was from BIO 101 Inc. (La Jolla, CA). The Subtractor I kit and PCR vectors were from Invitrogen (San Diego, CA). The 3` RACE system was from Life Technologies, Inc. (Burlington, Ontario). Phosphodiester oligodeoxynucleotides were synthesized on a 391 DNA synthesizer from Applied Biosystems (Foster City, CA). All other chemicals were from Sigma.

Isolation of Fetal Cells

Rats were sacrificed at 18, 19, 20, and 21 days of gestation (term = 22 days) by diethylether excess, and the fetuses were aseptically removed. The techniques used to prepare epithelial cell and fibroblast cultures have been described in detail previously(8) . Purity and viability of the cell cultures was of the same order (>90%) as reported previously (8) . Fetal lung cells were used within 24 h of isolation.

Glucocorticoid Treatment of Fetal Lung Fibroblasts

Day 18 and day 20 fetal lung fibroblasts were grown to confluence in Eagle's minimal essential medium (MEM) containing 2% (v/v) fetal bovine serum. At confluence, fibroblasts were rinsed twice with serum-free MEM, incubated for 24 h in serum-free MEM followed by another incubation of 24 h in fresh serum-free MEM with 10M cortisol. At the end of this incubation period, media was removed, and cells were washed and RNA was extracted.

Maternal Treatment of Fetal Lung with Glucocorticoids

On day 19 of gestation, pregnant rats were injected intraperitoneally with dexamethasone phosphate (200 µg/kg) (SABEX, Boucherville, Quebec). Controls were similarly injected with vehicle alone. Approximately 24 h after the injections, the fetuses were delivered by hysterotomy, killed, and their lungs isolated. The freshly, excised lungs were used for the preparation of fetal lung fibroblasts and epithelial cells as described previously(8) . RNA was extracted from these cells within 24 h of isolation.

Poly(A) RNA Purification

Total RNA was isolated by lysing the cells in 4 M guanidinium thiocyanate followed by centrifugation on a 5.7 M cesium chloride cushion to pellet RNA. After extraction with phenol/chloroform (1:1, v/v) the RNA was ethanol precipitated and collected by centrifugation. This RNA was air-dried and dissolved in RNase-free 10 mM Tris-HCl, 1 mM EDTA buffer, pH 7.4. Poly(A) mRNA was separated from total RNA using a mRNA purification kit. Briefly, 1.0 ml of total RNA (1.0-1.5 mg) was heat denatured at 65 °C for 5 min, followed by quick cooling on ice. After addition of 0.2 ml of sample buffer (10 mM Tris-HCl, 1 mM EDTA, 3.0 M NaCl, pH 7.4) the RNA sample was applied to an oligo(dT)-cellulose column which was pre-equilibrated with high salt buffer (10 mM Tris-HCl, 1 mM EDTA, 0.5 M NaCl, pH 7.4). The column was extensively washed with high salt buffer followed by low salt buffer (10 mM Tris-HCl, 1 mM EDTA, 0.1 M NaCl, pH 7.4). Poly(A) mRNA was eluted with 10 mM Tris-HCl, 1 mM EDTA, pH 7.4, which was prewarmed to 65 °C. A second identical column purification was performed to increase the purity of the poly(A) mRNA.

Construction of Full-length cDNA Library

Five µg of poly(A) mRNA, which was isolated from d20 fetal lung fibroblasts treated with 10M cortisol, was used to synthesize cDNA using a cDNA synthesis kit. Briefly, RNA was denatured and first strand cDNA synthesis was carried out at 37 °C in a reaction mixture containing Moloney murine leukemia virus reverse transcriptase, RNAguard, RNase- and DNase-free bovine serum albumin, oligo(dT) primer, and dNTPs. After a 1-h incubation, RNase H, DNA polymerase I, and dNTPs were added to the first stranded cDNA reaction to synthesize the second strand cDNA. This reaction was carried out at 12 °C for 1 h followed by another h at 22 °C. Following second strand synthesis, Klenow Fragment was added to blunt-end the cDNA. The reaction was terminated by heating at 65 °C for 10 min, followed by extraction of cDNA with phenol/chloroform (1:1, v/v) and purification on a Sephacryl S-300 spun column. Hemiphosphorylated EcoRI/NotI adaptors were ligated at 12 °C to blunt-ended cDNA with T4 DNA ligase. Following overnight incubation, the T4 ligase was denatured by heating at 65 °C, the EcoRI-terminated cDNA was phosphorylated with T4 polynucleotide kinase and then separated from unligated adapters on a second Sephacryl S-300 spun column. This processed cDNA was ligated into dephosphorylated ZAP II/EcoRI arms, packaged, and amplified following instructions provided by the supplier.

Construction of Enriched cDNA Library

Poly(A) RNA isolated from cortisol-treated fetal lung fibroblasts, subjacent to the epithelium(8) , was used to synthesize cDNA as described above. Following addition of EcoRI/NotI adaptors, the cDNA was size fractionated on a 1% (w/v) agarose gel. The cDNAs of 200-1000 bp were extracted from the gel using Geneclean, ligated into dephosphorylated gt10/EcoRI arms, packaged, and amplified following instructions provided by the supplier.

Preparation of Subtractive Probe

A subtractive probe to screen the cDNA libraries was generated using a subtractive hybridization technique involving photoactivatable biotin and phenol extraction. Briefly, poly(A) RNA (2 µg) of cortisol-treated fetal lung fibroblasts was reverse transcribed to cDNA in the presence of 100 µCi of [P]dCTP. The [P]-labeled cDNA was then hybridized for 48 h at 68 °C with excess (20 µg) photo-biotinylated mixed poly(A) RNA from d20 fetal (liver, kidney, and brain) and adult (lung) tissues and cortisol-treated d20 fetal skin fibroblasts. During this incubation period most sequences common to every organ and cortisol inducible in both fetal skin and lung fibroblasts hybridized. The resulting photobiotinylated RNAbulletcDNA hybrids were then complexed with free strepavidin. The strepavidin-photobiotinylated nucleic acid complex was removed from the hybridization mixture by selective phenol/chloroform (1:1, v/v) extraction which left unhybridized [P]cDNA behind. This [P]cDNA, which was highly enriched in sequences specific for cortisol-treated fetal lung fibroblasts, was then used to screen the cDNA libraries.

Screening of Enriched cDNA Library

Approximately 3-5 times 10^5 clones were screened with the subtracted [P]cDNA probe. The library was also screened with a [P]cDNA probe generated from mixed poly(A) RNA of fetal liver, kidney, brain, adult lung, and cortisol-treated fetal skin fibroblasts. Briefly, the library was titered and then plated with host cells on LB plates to approximately 2 times 10^4 plaques/plate. The phages were allowed to grow at 37 °C for 6-8 h. The plates were chilled at 4 °C, and plaques were transferred onto Hybond-N membranes. Duplicate filters were made. The filters were denatured with 0.5 M NaOH, neutralized, and baked at 80 °C for 2 h. The filters were prehybridized in 1.25 M NaCl, 0.25 M Tris, pH 7.4, 0.1 M NaK(2)HPO(4), 0.1 M EDTA, 50% (v/v) deionized formamide, 0.1% (w/v) SDS, 100 µg/µl boiled salmon sperm DNA overnight at 42 °C. The filters were then hybridized overnight at 42 °C in the same solution plus P-labeled probe (2-5 times 10^5 counts/min/filter). Following hybridization, filters washed twice at room temperature in 2 times SSC, 0.1% (w/v) SDS, then twice in 0.1 times SSC, 0.1% (w/v) SDS at 65 °C, air dried, and exposed to Kodak XAR-5 film at -80 °C with intensifying screens. Only clones positive for the subtracted cDNA probe and negative for the mixed cDNA probe were selected and rescreened using a newly generated subtractive cDNA probe. Second screening was performed at 100-fold lower dilution. Positive phages were grown on LB plates and phage DNA was purified according the plate lysate method(9) . The cDNA inserts were amplified by PCR using gt10 primers (5`-CTTTTGAGCAAGTTCAGCCTGGTTAAG-3` and 5`-GAGGTGGCTTATGAGTATTTCTTCCAGGGTA-3`. The PCR products were then directly ligated into a pCR vector with T4 DNA ligase. After transformation of competent Escherichia coli, positive colonies were picked for sequence analysis using the dideoxy chain termination method according to the manufacturer's instructions. DNA sequences were compared against sequences in the Genbank.

Screening of Full-length cDNA Library

Positive clones from the enriched cDNA library were then used to screen the full-lenghth cDNA library. After digestion of the pCR constructs with EcoRI, the cDNA inserts were separated by electrophoresis and extracted from the agarose gel using Geneclean. The cortisol-treated lung fibroblast cDNA library (ZAP II/EcoRI) was titered and plated with host cells on NZY plates at a density of 10^4 plaques/plate (total, 4-5 times 10^4 colonies). The plaques were transferred to nylon filters as described above. The filters were prehybridized in 0.8 M NaCl, 0.02 M PIPES, pH 6.5, 50% (v/v) deionized formamide, 0.5% (w/v) SDS, 100 µg/µl boiled salmon sperm DNA at 42 °C and then hybridized overnight at 42 °C in the same solution plus [P]cDNA probe (1 times 10^7 disintegrations/min/filter). Following hybridization, filters were washed with 2 times SSC and 0.2% (w/v) SDS at 42 °C for 30 min, then twice with 0.5 times SSC and 0.2% (w/v) SDS at 42 °C for 20 min, air dried, and exposed to Kodak XAR-5 film at -80 °C with intensifying screens. Second screening was performed at 100-fold lower dilution. After the secondary screening, positive colonies were picked up, and pBluescript plasmids were excised from positive ZAP phages according to the supplier's instructions.

Identification and Sequencing of TGFbeta(3)

One of the positive E. coli colonies (no. 12) was selected, grown in LB medium, and the plasmid was purified. The purified plasmid was digested with EcoRI, and the cDNA insert (size, 1833 bp) was separated by electrophoresis and extracted from the agarose gel using Geneclean. Following partial digestion with RsaI, the cDNA fragments were subcloned into pBluescript II KS+ vector (Stratagene, La Jolla, CA). Plasmids were prepared from selected clones, and inserts were sequenced using a T7 DNA Sequencing Kit.

Since this cDNA did not completely include the 3`-coding region and the 3`-untranslated region of the mRNA, 3` RACE (3`-Rapid Amplification of cDNA ends) was performed to amplify the 3` region of the mRNA to the poly(A) tract(10) . The 3` RACE products were cloned in the pCR vector and sequenced.

Northern Analysis

Total cellular RNA was isolated from fetal tissues and cells by lysing the cells in 4 M guanidinium thiocyanate followed by centrifugation on a 5.7 M cesium chloride cushion to pellet RNA. This total RNA (10 µg) was size fractionated on 1% agarose gel containing 3% (v/v) formaldehyde, transferred to Hybond N membranes, and immobilized by UV cross-linking. The TGFbeta cDNAs (TGFbeta(1) (985 kb), TGFbeta(2) (1037 bp), and TGFbeta(3) (no. 12, 304 bp)) were labeled with [alpha-P]dCTP using the random primer method. Prehybridization and hybridization were performed in 50% (v/v) formamide, 5 times SSPE, 0.5% (w/v) SDS, 5 times Denhardt's solution, and 100 µg/ml denatured salmon sperm DNA at 42 °C. Following hybridization, the blots were washed with 5 times SSC containing 0.2% (w/v) SDS at 42 °C followed by 2 times SSC with 0.2% (w/v) SDS at 42 °C and a final wash with 1 times SSC with 0.2% (w/v) SDS at 42 °C. The blots were autoradiographed with Kodak XAR-5 film overnight at -80 °C. Blots were then stripped and, for normalization, rehybridized with either a radiolabeled rat beta-actin (0.76 kb) or GAPDH cDNA (1.2 kb).

TGFbeta Bioassay

TGFbeta bioactivity was monitored as inhibition of the growth of mink lung epithelial cells, CCL-64 (American Tissue Culture Collection), using [^3H]thymidine incorporation assay mainly as described by Danielpour et al.(11) . The amount of TGFbeta was determined by comparison with a standard curve of standard amounts of either recombinant TGFbeta(1) or TGFbeta(3) (R & D Systems, Minneapolis, MN). Activity was identified as either TGFbeta(1) or TGFbeta(3) using neutralizing anti-TGFbeta(1) or anti-TGFbeta(3) antibodies (R & D Systems).

Effect of TGFbeta(3) on Fetal Lung Cell Proliferation and Differentiation

The effect of TGFbeta3 on fibroblast and epithelial cell proliferation was measured as described previously(8) . Briefly, quiescent cells of 19 days gestation were incubated in serum-free MEM containing 1 µCi/ml [^3H]thymidine and various concentrations of recombinant TGFbeta(3) (R & D Systems). After 18 h, the incubation was terminated, and thymidine incorporation into DNA was determined as described previously(8) . The effect of TGFbeta(3) on epithelial cell differentiation was assessed by incubating epithelial cells of 19 days gestation in serum-free MEM containing 1 µCi/ml [Me-^3H]choline and various concentrations of recombinant TGFbeta(3). After 24 h of incubation, the incorporation of radioactive choline into DSPC was determined as described previously(8) .


RESULTS

Cloning of Glucocorticoid-inducible Genes in Fetal Lung Fibroblasts

Based upon our previous findings that 1) fibroblast subjacent to the epithelium produced greater amounts of epithelial cell differentiation factors in response to cortisol then fibroblasts located some distance from the epithelium(8) , 2) maximal production of differentiation factors by fibroblasts in response to cortisol is observed with fibroblasts of 20-days gestation(8) , and 3) mRNA species of approximately 400 bp from cortisol-treated fetal lung fibroblasts translated in a cell-free system as well as in oocytes into a bioactive differentiation factor(5, 6, 7) , we synthesized cDNA from poly(A) RNA of cortisol-treated day 20 fetal lung fibroblasts, which were subjacent to the epithelium. The cDNA was fractionated by electrophoresis through an agarose gel and molecules between 200 and 1000 bp were recovered and inserted into gt10. This cDNA library, enriched in putative differentiation factors, was then hybridized with a subtractive [P]cDNA probe which was highly enriched for sequences specific for cortisol-treated fetal lung fibroblasts. Duplicate filters were hybridized with a mixed [P]cDNA from fetal (kidney, intestine, brain, and cortisol-treated skin fibroblasts) and adult (lung) tissues. Only clones positive for the subtractive probe and negative for the mixed cDNA were selected and rescreened. This screening strategy was based on previous observations that the production of differentiation factors by fetal lung fibroblasts in response to glucocorticoids was organ-specific (2) and gestation-dependent(8) . After the final screen, 57 positive clones were selected. One of the clones, no. 12 (304 bp), was randomly selected and characterized by sequencing. It showed a 89% sequence identity over 304 bp of the 3` end of the coding sequence of murine TGFbeta(3)(12) . Approximately 30% of the 57 positive clones hybridized with cDNA no. 12, implying that TGFbeta(3) is an important glucocorticoid-inducible gene in fetal lung fibroblasts. To further characterize rat TGFbeta(3), we used cDNA no. 12 as a probe to screen a full-length cDNA library of cortisol-treated fetal lung fibroblasts. Again, TGFbeta(3) seems to be highly expressed in these cells as approximately 0.05% of the total clones were positive. A clone, containing a 1833-bp cDNA insert, was grown up and the cDNA insert was isolated. The cDNA was partially digested with RsaI and subcloned for sequence analysis. The sequence analysis revealed that the cDNA did not include the complete 3`-coding region of message, which was then amplified and sequenced using 3` RACE. The rat lung TGFbeta(3) cDNA sequence is shown in Fig. 1. The coding region demonstrated 94% sequence similarity with TGFbeta(3) cDNA of mouse AKR-2B cells(12) . The putative amino acid sequence was altered at three positions when compared to murine TGFbeta(3).


Figure 1: Sequence of fetal rat lung fibroblast transforming growth factor-beta(3). Nucleotide sequence is shown with presumed reading frame. bullet, amino acids in presumed reading frame which are different from murine TGFbeta(3)(12) . Clone no. 12 is underlined.



Developmental Expression of TGFbeta(3) mRNA

Using cDNA no. 12 as a TGFbeta(3) probe, we found that the probe hybridized to a 3.9-kb mRNA species of whole fetal rat lung (Fig. 2). TGFbeta(3) mRNA levels were high in fetal lung tissue while weak expression was noted in other fetal tissues and adult lung. As can be seen in Fig. 3, TGFbeta(3) message was found in fetal lung fibroblasts but not in distal fetal lung epithelial cells. To determine whether the negative TGFbeta(3) mRNA expression in distal fetal lung epithelial cells was due to the culturing of the cells, we also examined TGFbeta(3) expression in fetal lung epithelial cells which were not allowed to adhere to the plastic. After the removal of fibroblasts by differential adherence, the non-adherent epithelial cells were collected, and RNA was extracted and analyzed for TGFbeta(3). No TGFbeta(3) transcripts were detected in these freshly isolated epithelial cells (not shown). The relative abundance of TGFbeta(3) mRNA levels in fetal lung fibroblasts increased after the pseudoglandular stage of lung development at 18 days of gestation and peaked during the early canalicular stage of lung development at 19 days of gestation, after which there was a decline in expression during the late canalicular stage (day 20) and the saccular stage (day 21) of gestation (Fig. 3). In order to determine whether all TGFbeta isoforms showed a similar developmental profile of gene expression, the membranes were rehybridized with TGFbeta(1) and TGFbeta(2) cDNAs. Message for TGFbeta(1) (2.3 kb) was also detected in fetal lung fibroblasts but not in fetal lung epithelial cells (Fig. 4). In contrast to TGFbeta(1) and TGFbeta(3), message for TGFbeta(2) (predominant mRNA band, 4.6 kb; minor band, 3.9 kb) was only detected in fetal lung epithelial cells (Fig. 4).


Figure 2: Expression of TGFbeta(3) mRNA in different fetal rat tissues and adult rat lung. Equal amounts of total RNA (10 µg), isolated from fetal lung, brain, liver, and intestine, and adult lungs, were electrophoresed on agarose gels, blotted, and the nylon membranes were hybridized as described under ``Experimental Procedures.''




Figure 3: Expression of TGFbeta(3) mRNA in fetal rat lung cels. Equal amounts of total RNA (10 µg), isolated from lung epithelial cells and fibroblasts of different gestational ages, were electrophoresed on agarose gels, blotted, and the nylon membranes were hybridized as described under ``Experimental Procedures.'' Equal RNA transfer was demonstrated by hybridizing the same blot with a GAPDH probe. The experiment was repeated twice with almost identical results.




Figure 4: Northern analysis for TGFbeta(1), -beta(2), and -beta(3) mRNAs in fetal rat lung cells. Equal amounts of total RNA (10 µg), isolated from lung epithelial cells and fibroblasts of different gestational ages, were electrophoresed on agarose gels, blotted, and the nylon membranes were successively hybridized with TGFbeta(1), -beta(2), and -beta(3) cDNAs as described under ``Experimental Procedures.'' Equal RNA transfer was demonstrated by hybridizing the same blot with a beta-actin probe. The TGFbeta(1) and -beta(3) probes hybridized with single mRNA transcripts of 3.9 and 2.3 kb, respectively. The TGFbeta(2) cDNA hybridized to two mRNA sizes of 4.6 and 3.9 kb. The experiment was repeated with almost identical results.



Glucocorticoid Effect on Expression of TGFbeta(3) mRNA

To examine the effect of glucocorticoids on TGFbeta(3) mRNA expression, day 18 fetal lung fibroblasts were exposed for various times (0-48 h) to different concentrations of cortisol. As can be seen in Fig. 5, cortisol increased the expression of TGFbeta(3) mRNA in fetal lung fibroblasts in a time- and concentration-dependent manner. Maximal expression was noted with 10M cortisol after 24 h. Cortisol treatment did not increase the mRNA expression for TGFbeta(1) in fetal lung fibroblasts (mRNA level (treated/control) = 0.89 ± 0.05, mean ± S.E., n = 3 for 10M cortisol), suggesting that glucocorticoids specifically induce TGFbeta(3) gene expression. Cortisol did not induce TGFbeta(3) mRNA expression in fetal intestinal and skin fibroblasts (not shown), implying that the glucocorticoid induction of TGFbeta(3) is organ-specific. To determine whether glucocorticoids affect the expression of TGFbeta(3) mRNA in vivo, we administered dexamethasone to pregnant rats at 18 days of gestation and isolated 24 h later the fetal lung fibroblasts and epithelial cells. Maternal administration of dexamethasone increased the TGFbeta(3) mRNA levels in fetal lung fibroblasts without inducing TGFbeta(3) expression in fetal epithelial cells (Fig. 6). This suggests that the glucocorticoid induction of TGFbeta(3) is cell type-specific.


Figure 5: Effect of time and dose of cortisol exposure on TGFbeta(3) mRNA expression in fetal rat lung fibroblasts. Left panel, day 18 fetal lung fibroblasts were cultured in presence of 10M cortisol for 0-48 h. Total RNA (10 µg) was hybridized to P-labeled TGFbeta(3) probe, and the intensity of the specific mRNA band was quantitated by laser densitometry. Each time point represents mean of two to three experiments. Right panel, day 18 fibroblasts were cultured in presence of various concentrations of cortisol for 24 h. Total RNA (10 µg) was hybridized to P-labeled TGFbeta(3) probe, and the intensity of the specific mRNA band was quantitated by laser densitometry. Each point represents means ± S.E. of three experiments.




Figure 6: Effect of maternal administration of dexamethasone on TGFbeta(3) mRNA expression in fetal rat lung cells. Day 19 pregnant rats were injected with 200 µg/kg dexamethasone or vehicle alone. Approximately 24 h after the injections, the fetuses were delivered, and fetal lung fetal lung fibroblasts and epithelial cells were isolated. Within 24 h of isolation, total RNA was extracted, electrophoresed on agarose gels, blotted, and the nylon membranes were hybridized with a P-labeled TGFbeta(3) cDNA. Equal RNA transfer was demonstrated by hybridizing the same blot with a beta-actin probe.



Autoregulation of TGFbeta(3) mRNA Expression

Several studies have suggested that TGFbeta up-regulates its own gene expression (13, 14) . In order to investigate whether exogenous TGFbeta(3) affected TGFbeta(3) gene expression, fetal lung fibroblasts were incubated for 24 h with 0-10 ng/ml TGFbeta(3), following which TGFbeta(3) mRNA was measured by Northern hybridization. As shown in Fig. 7, exposure of fetal lung fibroblasts to increasing concentrations of exogenous TGFbeta(3) resulted in a concentration-dependent down-regulation of TGFbeta(3) mRNA expression.


Figure 7: Effect of exogenous TGFbeta(3) on TGFbeta(3) mRNA expression in fetal rat lung fibroblasts. Day 19 fibroblasts were exposed to 0-10 ng/ml recombinant TGFbeta(3) for 24 h, following which total RNA was isolated. Total RNA (10 µg) was hybridized to P-labeled TGFbeta(3) and beta-actin probes and the intensity of the specific mRNA bands was quantitated by laser densitometry. Results are expressed as a ratio over beta-actin. Each point represents mean ± S.E. of three experiments. *, significantly different (p > 0.05) from controls.



Glucocorticoid Effect on TGFbeta(3) Bioactivity

Bioassays of conditioned media from the cells showed that fetal lung fibroblasts secrete TGFbeta activity into their medium and that cortisol exposure increased the TGFbeta activity in the medium by 64% (Table 1). Approximately 80% of the TGFbeta activity in the conditioned medium was found to be in the active form. Cortisol treatment did not alter the amount of activated TGFbeta. Total TGFbeta activity increased by 104% after cortisol treatment. This suggests that the cortisol-induced increase in TGFbeta activity is not due to an increased conversion of latent TGFbeta to active TGFbeta but most likely due to an increase in TGFbeta protein synthesis. Addition of a neutralizing TGFbeta(3) antibody showed that 20% of the activity in media conditioned by fetal lung fibroblasts in the absence of cortisol was due to TGFbeta(3). After cortisol exposure, 70% of the total TGFbeta activity in the media was due to TGFbeta(3), suggesting that cortisol specifically increased the expression of TGFbeta(3) in fetal lung fibroblasts.



Effect of TGFbeta3 on Fetal Lung Cell Proliferation and Differentiation

TGFbeta is expressed in many tissues including lung. It has multiple effects on cellular proliferation. TGFbeta inhibits proliferation of certain cells and augments that of others (13) . To determine the effect of TGFbeta(3) fetal lung cell growth, fibroblasts and epithelial cells were incubated with recombinant TGFbeta(3), and its influence on DNA synthesis was measured. Independent of dosage used, TGFbeta(3) had no effect on cell proliferation (Table 2). Medium conditioned by fibroblast in the presence of glucocorticoids (FCM) has been shown to stimulate synthesis of the major surfactant lipid, DSPC, by distal fetal lung epithelial cells(1, 2, 3, 4) . To examine whether TGFbeta(3) could mimic the stimulatory effect of FCM on surfactant lipid synthesis, and by inference, epithelial differentiation, epithelial cells were incubated with recombinant TGFbeta(3) and DSPC synthesis was measured. Addition of 0-40 ng/ml TGFbeta(3) to the epithelial cell cultures failed to stimulate epithelial cell differentiation (Table 2).




DISCUSSION

Using subtractive hybridization, we cloned and identified TGFbeta(3) as a glucocorticoid-inducible gene in fetal rat lung fibroblasts. We found that glucocorticoids induced TGFbeta(3) expression in a dose-dependent manner in cultured fetal rat lung fibroblasts. Under similar experimental conditions as used for fetal rat lung fibroblasts, glucocorticoids did not induce TGFbeta(3) mRNA expression in fetal rat skin and intestinal fibroblasts. Treatment of fetal lung fibroblasts with glucocorticoids also did not affect TGFbeta(1) mRNA expression. Thus, although cell culture findings should be interpreted with caution, these data are compatible with TGFbeta(3) playing an important role in late fetal lung development. A functional role for TGFbeta(3) in lung maturation is supported by several other findings. First, we found that message for TGFbeta(3) was far more abundant in fetal lung than in other fetal tissues and adult lung. Second, maximal TGFbeta(3) mRNA expression in fetal lung fibroblasts occurred around the time when circulating glucocorticoids are rising(2) . Third, maximal expression of TGFbeta(3) coincided with that of the glucocorticoid receptor in fetal lung fibroblasts(4) . As endogenous glucocorticoids and glucocorticoid receptors are known to play a physiological role in lung maturation(2) , it is tempting to speculate that endogenous glucocorticoids in part mediate their effect on lung maturation via TGFbeta(3). The finding that maternal administration of glucocorticoids increased TGFbeta(3) mRNA expression in fetal lung fibroblasts supports this concept. The exact function of TGFbeta(3) remains to be determined, but it has been implicated in cell growth and extracellular matrix remodelling (13) . In the present study, we found that neither fibroblast nor epithelial cell proliferation were affected by TGFbeta(3). In contrast, we have previously reported that very low dosages of TGFbeta(1) stimulated distal fetal lung epithelial cell growth (8) . In addition, we found that fibroblasts from the pseudoglandular (day 18) and early canalicular (day 19) stages of lung development stimulated epithelial cell proliferation(8) . Using quantitative reverse transcriptase-PCR for TGFbeta(1)(15) , expression of TGFbeta(1) by fetal lung fibroblasts was maximal at day 18 and decreased with advancing gestation (not shown). The maximal TGFbeta(1) expression by fibroblasts during the pseudoglandular period agrees with TGFbeta(1) being a stimulatory mitogen for epithelial cell growth during this period of lung development. We have also shown that neutralizing TGFbeta antibodies stimulated fetal lung epithelial cell proliferation(8) . We speculated that epithelial cells released a TGFbeta-like activity which acted in an autocrine fashion to regulate epithelial cell growth. The present data suggest that this putative TGFbeta-like activity is composed entirely of the TGFbeta(2) isoform. TGFbeta has also been implicated as an important regulator of differentiation of epithelial cells. It has been shown that TGFbeta induces squamous differentiation of bronchial (16) and tracheal (17) cells. Previously, we and others showed that increasing concentrations of TGFbeta(1) inhibit differentiation of cultured distal fetal lung epithelial cells(8, 18, 19) . The present study showed that increasing concentrations of TGFbeta(3) did not affect distal fetal lung epithelial cell differentiation, assessed as DSPC synthesis, suggesting that TGFbeta(3) is not the fibroblast-derived maturation factor, fibroblast pneumocyte factor (FPF)(1, 2, 3, 4) .

The cloning of TGFbeta(3) from rat lung fibroblasts was an unexpected finding and raises some questions regarding the cloning procedure. We originally screened a library enriched for short cDNAs as we have previously reported that a putative epithelial cell differentiation factor (FPF) released by fetal lung fibroblasts in response to glucocorticoids is encoded by mRNA species of approximately 400 bp(5, 6, 7) . However, the TGFbeta(3) mRNA species in fetal lung fibroblasts is around 3.9 kb. The presence of short TGFbeta(3) cDNAs in this enriched library may be due to degradation of TGFbeta(3) message during the mRNA isolation or incomplete cDNA synthesis. It is also possible that TGFbeta(3) mRNA is rapidly degraded in the cells. To date, no studies have compared the mRNA stabilities of the different TGFbeta forms. Our observation that the TGFbeta(3) probe did not hybridize with smaller TGFbeta(3) transcripts makes it unlikely that TGFbeta(3) is rapidly degraded.

TGFbeta expression has been described during fetal mouse lung development. In situ hybrization studies (20, 21) have demonstrated prominent expression of TGFbeta(1) mRNA throughout the mesenchyme, in agreement with our Northern analysis. TGFbeta(2) mRNA has been shown to localize mainly to the epithelium of the developing distal airways, which is also consistent with our findings. The TGFbeta(3) mRNA expression pattern changed during lung development. Initially, transcripts were predominantly found in the tracheal mesenchyme, but TGFbeta(3) signals were visible in the epithelium of the growing bronchioles during the pseudoglandular stage (day 14.5) of murine lung development. No transcripts were detected by day 16.5. Unfortunately, Schmid et al.(21) did not assess TGFbeta(3) expression during the later stages of fetal murine lung development (day 17-18, term = 19 days). Our data show that TGFbeta(3) mRNA is transiently expressed in mesenchymal cells during the canalicular stage (day 19-20) of rat lung development. Recent studies suggest that TGFbeta expression depends on tissue architecture. Autoinduction of TGFbeta expression in tumor cells cultured in a two-dimensional monolayer is different from tumor cells grown as in vivo-like three-dimensional speroids(22) . Thus, it is possible that the negative TGFbeta(3) expression in distal fetal lung epithelial cells is a culture phenomenon. Another possibility is that TGFbeta(3) mRNA expression by epithelial cells is influenced by mesenchymal-epithelial interactions. However, freshly isolated fetal lung epithelial cells, which were not allowed to adhere to the plastic and were not cultured for 24 h, also did not express TGFbeta(3) mRNA. This strongly suggests that TGFbeta(3) is not expressed by distal fetal lung epithelial cells in situ.

In the present study, we demonstrate that fetal rat lung fibroblasts down-regulate their TGFbeta(3) gene expression in response to exogenous TGFbeta(3). Autoinduction of TGFbeta expression has been reported for TGFbeta(1) in human fetal and adult lung fibroblast cell lines(15) . Murine embryo AKR-2B fibroblasts have been shown to down-regulate TGFbeta(1) expression in response to TGFbeta(3)(23) . It is possible that the developmental decrease in TGFbeta(1) expression by fetal lung fibroblasts is due to a negative autocrine regulation by TGFbeta(3). However, our observation that glucocorticoids induce TGFbeta(3) but not TGFbeta(1) mRNA expression suggests that TGFbeta(1) gene expression is not affected by TGFbeta(3).

It has been shown that fetal rat lung fibroblasts produce TGFbeta molecules, but the identity of the TGFbeta forms has not been studied (24) . TGFbetas are mainly secreted in biologically inactive forms(14, 25) . Activation occurs in vitro by a variety of nonphysiological treatments, such as heating or exposure to low pH(26, 27) . Although the activation mechanism in vivo remains unknown, proteases and glycosidases have been implicated in activating latent TGFbeta(27, 28) . Our data suggest that TGFbeta released by fetal rat lung fibroblasts is preactivated. This is likely due to prompt proteolytic processing. Unlike our primary cells, rat lung cell lines have been shown to secrete mainly latent TGFbeta(24) .

The role of TGFbeta in late fetal lung development is not understood. Torday and Kourembanas (19) reported that immature rat lung fibroblasts at days 15-19 of fetal gestation produce a TGFbeta-like activity which antagonizes the maturation factor (FPF) produced by mature rat lung fibroblasts at days 20-21 of fetal gestation in response to glucocorticoids. Our ontogeny data for TGFbeta(1) are compatible with this observation. Recently, Nielsen et al. (29) showed that TGFbeta(1) interfered with the capability of the distal fetal lung epithelial cell to respond to FPF. Since both FPF and TGFbeta(3) are induced by glucocorticoids in fetal lung fibroblasts, it is unlikely that TGFbeta(3) has a similar function. It is apparent that further studies are necessary to elucidate the functional role of TGFbeta(3) in fetal lung maturation.


FOOTNOTES

*
These studies were supported by a group grant from the Medical Research Council of Canada and an equipment grant from the Ontario Thoracic Society. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U03491[GenBank].

§
To whom correspondence should be addressed: Div. Neonatology, Hospital for Sick Children, 555 University Ave., Toronto, Ontario, M5G 1X8, Canada. Tel.: 416-598-6772; Fax: 416-813-5002.

(^1)
The abbreviations used are: TGFbeta(3), transforming growth factor-beta(3); bp, base pair(s); GAPDH, glyceraldehyde-3-phosphate dehydrogenase; kb, kilobase(s); PCR, polymerase chain reaction; MEM, Eagle's minimal essential medium; PIPES, 1,4-piperazinediethanesulfonic acid; DSPC, disaturated phosphatidylcholine; FPF, fibroblast pneumocyte factor.


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