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 TGF
(
)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
TGF
1 (insert size, 983 bp) and mouse TGF
2 (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
-actin cDNA (insert size 0.76 kb) was generated by
reverse transcriptase-PCR cloning using rat
-actin primers
(Clontech, Palo Alto, CA). Hybond N
membranes,
[
-
P]dCTP,
[
H]thymidine, and
[Me-
H]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
10
M 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 10
M 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 RNA
cDNA
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
10
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
10
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
HPO
,
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
10
counts/min/filter). Following hybridization, filters washed twice
at room temperature in 2
SSC, 0.1% (w/v) SDS, then twice in 0.1
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
plaques/plate (total, 4-5
10
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
10
disintegrations/min/filter). Following hybridization, filters
were washed with 2
SSC and 0.2% (w/v) SDS at 42 °C for 30
min, then twice with 0.5
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
TGF

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 TGF
cDNAs (TGF
(985 kb),
TGF
(1037 bp), and TGF
(no. 12, 304
bp)) were labeled with [
-
P]dCTP using the
random primer method. Prehybridization and hybridization were performed
in 50% (v/v) formamide, 5
SSPE, 0.5% (w/v) SDS, 5
Denhardt's solution, and 100 µg/ml denatured salmon sperm DNA
at 42 °C. Following hybridization, the blots were washed with 5
SSC containing 0.2% (w/v) SDS at 42 °C followed by 2
SSC with 0.2% (w/v) SDS at 42 °C and a final wash with 1
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
-actin (0.76 kb) or GAPDH cDNA (1.2 kb).
TGF
Bioassay
TGF
bioactivity was
monitored as inhibition of the growth of mink lung epithelial cells,
CCL-64 (American Tissue Culture Collection), using
[
H]thymidine incorporation assay mainly as
described by Danielpour et al.(11) . The amount of
TGF
was determined by comparison with a standard curve of standard
amounts of either recombinant TGF
or TGF
(R & D Systems, Minneapolis, MN). Activity was identified as
either TGF
or TGF
using neutralizing
anti-TGF
or anti-TGF
antibodies (R
& D Systems).
Effect of TGF
on Fetal Lung Cell
Proliferation and Differentiation
The effect of TGF
3 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
[
H]thymidine and various concentrations of
recombinant TGF
(R & D Systems). After 18 h, the
incubation was terminated, and thymidine incorporation into DNA was
determined as described previously(8) . The effect of
TGF
on epithelial cell differentiation was assessed by
incubating epithelial cells of 19 days gestation in serum-free MEM
containing 1 µCi/ml [Me-
H]choline
and various concentrations of recombinant TGF
. 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 TGF
(12) .
Approximately 30% of the 57 positive clones hybridized with cDNA no.
12, implying that TGF
is an important
glucocorticoid-inducible gene in fetal lung fibroblasts. To further
characterize rat TGF
, we used cDNA no. 12 as a probe
to screen a full-length cDNA library of cortisol-treated fetal lung
fibroblasts. Again, TGF
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 TGF
cDNA sequence is shown in Fig. 1. The coding region demonstrated 94% sequence similarity
with TGF
cDNA of mouse AKR-2B cells(12) . The
putative amino acid sequence was altered at three positions when
compared to murine TGF
.
Figure 1:
Sequence of fetal rat lung fibroblast
transforming growth factor-
. Nucleotide sequence is
shown with presumed reading frame.
, amino acids in presumed
reading frame which are different from murine
TGF
(12) . Clone no. 12 is underlined.
Developmental Expression of TGF
mRNA
Using cDNA no. 12 as a TGF
probe, we
found that the probe hybridized to a 3.9-kb mRNA species of whole fetal
rat lung (Fig. 2). TGF
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, TGF
message was found in fetal lung fibroblasts but not in distal
fetal lung epithelial cells. To determine whether the negative
TGF
mRNA expression in distal fetal lung epithelial
cells was due to the culturing of the cells, we also examined
TGF
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
TGF
. No TGF
transcripts were detected
in these freshly isolated epithelial cells (not shown). The relative
abundance of TGF
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 TGF
isoforms showed a similar developmental profile of
gene expression, the membranes were rehybridized with TGF
and TGF
cDNAs. Message for TGF
(2.3 kb) was also detected in fetal lung fibroblasts but not in
fetal lung epithelial cells (Fig. 4). In contrast to
TGF
and TGF
, message for
TGF
(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 TGF
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 TGF
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
TGF
, -
, and -
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
TGF
, -
, and -
cDNAs
as described under ``Experimental Procedures.'' Equal RNA
transfer was demonstrated by hybridizing the same blot with a
-actin probe. The TGF
and -
probes hybridized with single mRNA transcripts of 3.9 and 2.3 kb,
respectively. The TGF
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 TGF
mRNA
To examine the effect of glucocorticoids on
TGF
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 TGF
mRNA in fetal lung
fibroblasts in a time- and concentration-dependent manner. Maximal
expression was noted with 10
M cortisol
after 24 h. Cortisol treatment did not increase the mRNA expression for
TGF
in fetal lung fibroblasts (mRNA level
(treated/control) = 0.89 ± 0.05, mean ± S.E., n = 3 for 10
M cortisol),
suggesting that glucocorticoids specifically induce TGF
gene expression. Cortisol did not induce TGF
mRNA expression in fetal intestinal and skin fibroblasts (not
shown), implying that the glucocorticoid induction of TGF
is organ-specific. To determine whether glucocorticoids affect
the expression of TGF
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 TGF
mRNA levels in fetal lung fibroblasts without inducing
TGF
expression in fetal epithelial cells (Fig. 6). This suggests that the glucocorticoid induction of
TGF
is cell type-specific.
Figure 5:
Effect of time and dose of cortisol
exposure on TGF
mRNA expression in fetal rat lung
fibroblasts. Left panel, day 18 fetal lung fibroblasts were
cultured in presence of 10
M cortisol for
0-48 h. Total RNA (10 µg) was hybridized to
P-labeled TGF
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 TGF
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 TGF
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 TGF
cDNA. Equal RNA transfer
was demonstrated by hybridizing the same blot with a
-actin probe.
Autoregulation of TGF
mRNA
Expression
Several studies have suggested that TGF
up-regulates its own gene expression (13, 14) . In
order to investigate whether exogenous TGF
affected
TGF
gene expression, fetal lung fibroblasts were
incubated for 24 h with 0-10 ng/ml TGF
,
following which TGF
mRNA was measured by Northern
hybridization. As shown in Fig. 7, exposure of fetal lung
fibroblasts to increasing concentrations of exogenous TGF
resulted in a concentration-dependent down-regulation of
TGF
mRNA expression.
Figure 7:
Effect of
exogenous TGF
on TGF
mRNA expression
in fetal rat lung fibroblasts. Day 19 fibroblasts were exposed to
0-10 ng/ml recombinant TGF
for 24 h, following
which total RNA was isolated. Total RNA (10 µg) was hybridized to
P-labeled TGF
and
-actin probes and
the intensity of the specific mRNA bands was quantitated by laser
densitometry. Results are expressed as a ratio over
-actin. Each
point represents mean ± S.E. of three experiments. *,
significantly different (p > 0.05) from
controls.
Glucocorticoid Effect on TGF
Bioactivity
Bioassays of conditioned media from the cells
showed that fetal lung fibroblasts secrete TGF
activity into their
medium and that cortisol exposure increased the TGF
activity in
the medium by 64% (Table 1). Approximately 80% of the TGF
activity in the conditioned medium was found to be in the active form.
Cortisol treatment did not alter the amount of activated TGF
.
Total TGF
activity increased by 104% after cortisol treatment.
This suggests that the cortisol-induced increase in TGF
activity
is not due to an increased conversion of latent TGF
to active
TGF
but most likely due to an increase in TGF
protein
synthesis. Addition of a neutralizing TGF
antibody
showed that 20% of the activity in media conditioned by fetal lung
fibroblasts in the absence of cortisol was due to TGF
.
After cortisol exposure, 70% of the total TGF
activity in the
media was due to TGF
, suggesting that cortisol
specifically increased the expression of TGF
in fetal
lung fibroblasts.
Effect of TGF
3 on Fetal Lung Cell Proliferation and
Differentiation
TGF
is expressed in many tissues including
lung. It has multiple effects on cellular proliferation. TGF
inhibits proliferation of certain cells and augments that of others (13) . To determine the effect of TGF
fetal
lung cell growth, fibroblasts and epithelial cells were incubated with
recombinant TGF
, and its influence on DNA synthesis
was measured. Independent of dosage used, TGF
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 TGF
could mimic the stimulatory effect of FCM
on surfactant lipid synthesis, and by inference, epithelial
differentiation, epithelial cells were incubated with recombinant
TGF
and DSPC synthesis was measured. Addition of
0-40 ng/ml TGF
to the epithelial cell cultures
failed to stimulate epithelial cell differentiation (Table 2).
DISCUSSION
Using subtractive hybridization, we cloned and identified
TGF
as a glucocorticoid-inducible gene in fetal rat
lung fibroblasts. We found that glucocorticoids induced TGF
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 TGF
mRNA expression in fetal rat skin and intestinal fibroblasts.
Treatment of fetal lung fibroblasts with glucocorticoids also did not
affect TGF
mRNA expression. Thus, although cell
culture findings should be interpreted with caution, these data are
compatible with TGF
playing an important role in late
fetal lung development. A functional role for TGF
in
lung maturation is supported by several other findings. First, we found
that message for TGF
was far more abundant in fetal
lung than in other fetal tissues and adult lung. Second, maximal
TGF
mRNA expression in fetal lung fibroblasts occurred
around the time when circulating glucocorticoids are
rising(2) . Third, maximal expression of TGF
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
TGF
. The finding that maternal administration of
glucocorticoids increased TGF
mRNA expression in fetal
lung fibroblasts supports this concept. The exact function of
TGF
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
TGF
. In contrast, we have previously reported that
very low dosages of TGF
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 TGF
(15) , expression of TGF
by fetal lung fibroblasts was maximal at day 18 and decreased
with advancing gestation (not shown). The maximal TGF
expression by fibroblasts during the pseudoglandular period
agrees with TGF
being a stimulatory mitogen for
epithelial cell growth during this period of lung development. We have
also shown that neutralizing TGF
antibodies stimulated fetal lung
epithelial cell proliferation(8) . We speculated that
epithelial cells released a TGF
-like activity which acted in an
autocrine fashion to regulate epithelial cell growth. The present data
suggest that this putative TGF
-like activity is composed entirely
of the TGF
isoform. TGF
has also been implicated
as an important regulator of differentiation of epithelial cells. It
has been shown that TGF
induces squamous differentiation of
bronchial (16) and tracheal (17) cells. Previously, we
and others showed that increasing concentrations of TGF
inhibit differentiation of cultured distal fetal lung epithelial
cells(8, 18, 19) . The present study showed
that increasing concentrations of TGF
did not affect
distal fetal lung epithelial cell differentiation, assessed as DSPC
synthesis, suggesting that TGF
is not the
fibroblast-derived maturation factor, fibroblast pneumocyte factor
(FPF)(1, 2, 3, 4) .
The cloning
of TGF
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 TGF
mRNA species in fetal lung fibroblasts is around 3.9 kb. The
presence of short TGF
cDNAs in this enriched library
may be due to degradation of TGF
message during the
mRNA isolation or incomplete cDNA synthesis. It is also possible that
TGF
mRNA is rapidly degraded in the cells. To date, no
studies have compared the mRNA stabilities of the different TGF
forms. Our observation that the TGF
probe did not
hybridize with smaller TGF
transcripts makes it
unlikely that TGF
is rapidly degraded.
TGF
expression has been described during fetal mouse lung development. In situ hybrization studies (20, 21) have
demonstrated prominent expression of TGF
mRNA
throughout the mesenchyme, in agreement with our Northern analysis.
TGF
mRNA has been shown to localize mainly to the
epithelium of the developing distal airways, which is also consistent
with our findings. The TGF
mRNA expression pattern
changed during lung development. Initially, transcripts were
predominantly found in the tracheal mesenchyme, but TGF
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 TGF
expression
during the later stages of fetal murine lung development (day
17-18, term = 19 days). Our data show that TGF
mRNA is transiently expressed in mesenchymal cells during the
canalicular stage (day 19-20) of rat lung development. Recent
studies suggest that TGF
expression depends on tissue
architecture. Autoinduction of TGF
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 TGF
expression
in distal fetal lung epithelial cells is a culture phenomenon. Another
possibility is that TGF
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 TGF
mRNA. This strongly suggests that
TGF
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 TGF
gene expression in response to exogenous TGF
.
Autoinduction of TGF
expression has been reported for
TGF
in human fetal and adult lung fibroblast cell
lines(15) . Murine embryo AKR-2B fibroblasts have been shown to
down-regulate TGF
expression in response to
TGF
(23) . It is possible that the
developmental decrease in TGF
expression by fetal lung
fibroblasts is due to a negative autocrine regulation by
TGF
. However, our observation that glucocorticoids
induce TGF
but not TGF
mRNA
expression suggests that TGF
gene expression is not
affected by TGF
.
It has been shown that fetal rat
lung fibroblasts produce TGF
molecules, but the identity of the
TGF
forms has not been studied (24) . TGF
s 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 TGF
(27, 28) . Our
data suggest that TGF
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 TGF
(24) .
The role of TGF
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 TGF
-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 TGF
are
compatible with this observation. Recently, Nielsen et al. (29) showed that TGF
interfered with the
capability of the distal fetal lung epithelial cell to respond to FPF.
Since both FPF and TGF
are induced by glucocorticoids
in fetal lung fibroblasts, it is unlikely that TGF
has
a similar function. It is apparent that further studies are necessary
to elucidate the functional role of TGF
in fetal lung
maturation.