1 Department of Medicine and 3 Department of Pediatrics, Duke University Medical Center, and 2 Research Service, Durham Veterans Affairs Medical Center, Durham, North Carolina 27710
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ABSTRACT |
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Transforming growth factor (TGF)- is
a family of multifunctional cytokines controlling cell growth,
differentiation, and extracellular matrix deposition in the lung. The
biological effects of TGF-
are mediated by type I (T
R-I) and II
(T
R-II) receptors. Our previous studies show that the expression of
T
R-II is highly regulated in a spatial and temporal fashion during
lung development. In the present studies, we investigated the
temporal-spatial pattern and cellular expression of T
R-I during lung
development. The expression level of T
R-I mRNA in rat lung at
different embryonic and postnatal stages was analyzed by Northern
blotting. T
R-I mRNA was expressed in fetal rat lungs in early
development and then decreased as development proceeded. The
localization of T
R-I in fetal and postnatal rat lung tissues was
investigated by using in situ hybridization performed with an antisense
RNA probe. T
R-I mRNA was present in the mesenchyme and epithelium of
gestational day 14 rat lungs. An intense T
R-I signal was
observed in the epithelial lining of the developing bronchi. In
gestational day 16 lungs, the expression of T
R-I mRNA was
increased in the mesenchymal tissue. The epithelium in both the distal
and proximal bronchioles showed a similar level of T
R-I expression.
In postnatal lungs, T
R-I mRNA was detected in parenchymal tissues
and blood vessels. We further studied the expression of T
R-I in
cultured rat lung cells. T
R-I was expressed by cultured rat lung
fibroblasts, microvascular endothelial cells, and alveolar epithelial
cells. These studies demonstrate a differential regulation and
localization of T
R-I that is different from that of T
R-II during
lung development. T
R-I, T
R-II, and TGF-
isoforms exhibit
distinct but overlapping patterns of expression during lung
development. This implies a distinct role for T
R-I in mediating
TGF-
signal transduction during lung development.
transforming growth factor-; mesenchyme; epithelium
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INTRODUCTION |
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TRANSFORMING GROWTH FACTOR (TGF)- is a family of
multifunctional cytokines that is involved in controlling many
biological activities including cell growth, differentiation,
extracellular matrix deposition, and cell cycle progression in a
variety of cell types (14, 19). The biological effects of TGF-
are
known to be exerted on cells through interaction of the ligands with their specific transmembrane receptors. A number of different types of
putative receptors for TGF-
, including three distinct size classes
termed type I (T
R-I; 50-60 kDa), type II (T
R-II; 75-85
kDa), and type III (T
R-III; a 280-kDa proteoglycan with a 120-kDa
core protein), have been identified by affinity cross-linking experiments (3, 20). Sequence analysis of T
R-III revealed that it is
a transmembrane proteoglycan with a short and highly conserved
cytoplasmic domain that has no apparent signal motif (17, 34). T
R-I
and T
R-II are transmembrane serine/threonine kinases that consist of
a short extracellular domain, a single transmembrane region, and a
kinase domain (9, 16). Although T
R-I and T
R-II share similar
domain structures, T
R-I distinguishes itself from T
R-II by
several features. T
R-I has a unique highly conserved GS motif
(SGSGSG) in the juxtamembrane region immediately preceding the kinase
domain. Ligand-induced phosphorylation of the serines and threonines in
the GS domain is required for activation of signaling (31). After the
GS sequence, T
R-I has an LP motif. The immunophilin 12-kDa FK506
binding protein binds the LP sequence of T
R-I, inhibiting TGF-
signaling (5). T
R-I also has a short extracellular domain and
essentially no COOH-terminal extension after the kinase domain.
Both TR-I and T
R-II are indispensable for TGF-
signaling (2,
9, 16). T
R-II is capable of binding TGF-
independently, but
binding of T
R-I to TGF-
requires the presence of T
R-II. Once
T
R-II binds to TGF-
, it results in the recruitment of T
R-I to
form a heteromeric complex. Activated T
R-II transphosphorylates T
R-I kinase, thereby activating T
R-I, which then propagates the
signal to downstream substrates (33, 36). Recent studies (8, 15)
revealed that Mothers against dpp (Mad) in Drosophila and its
homologs play important roles in the intracellular signal transduction
of the serine/threonine kinase receptors. TGF-
induces heteromeric
complexes of Smad2, Smad3, and Smad4 and their concomitant translocation to the nucleus, which is required for TGF-
signal transduction (21, 25).
Lung branching morphogenesis and alveolarization are a complex process
of reciprocal interactions between epithelial and mesenchymal tissue
that require precise regulatory control. A regulatory role for TGF-
in the developing lung has been implicated because the TGF-
family
is composed of highly conserved cytokines with demonstrated pleiotropic
effects on their own biosynthesis, mesenchymal mitogenesis, and
epithelial differentiation. T
R-II and T
R-I have been shown to be
required for growth regulation and extracellular matrix production by
TGF-
in lung fibroblasts (39, 41). Until the question of cellular
localization of its receptors is resolved, the mechanism by which
TGF-
may regulate lung development will remain speculative. A
previous study by Zhao and Young (40) showed that
expression of the T
R-II is temporally and spatially regulated during
rat lung development (40). In an effort to gain further clues about the
function and mechanism of TGF-
signaling during lung development, we
examined the temporal expression of T
R-I by Northern blot analysis
and the spatial localization of T
R-I mRNA by in situ hybridization
of rat embryo and rat lung tissue. T
R-I mRNA shows a distinctive
developmental localization and is highly regulated during lung
development. These studies provide direct evidence for differential
expression and localization of T
R-I compared with T
R-II during
rat lung development.
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MATERIALS AND METHODS |
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Animals and tissue preparation.
Timed-pregnant Sprague-Dawley rats were purchased from Zivic Miller
(Allison Park, PA). At least two and as many as five separate litters
of donor animals or adults (7-8 wk of age) were used for analysis
at each time point. The lungs were removed while the animals were under
pentobarbital sodium (50 mg/kg) anesthesia, frozen in liquid nitrogen,
and stored at 70°C. Lung tissues were fixed in ice-cold 4%
paraformaldehyde in phosphate-buffered saline (PBS), dehydrated through
graded ethanol, and embedded in paraffin. Five-micrometer-thick
sections were cut and mounted on 3-aminopropyltriethoxysilane (Sigma,
St. Louis, MO)-treated glass slides.
Cell lines and culture techniques. Fetal rat lung fibroblasts were isolated from gestational day 16 fetal rat lung tissue by culture of tissue explants in Dulbecco's modified Eagle's medium (DMEM) and 10% fetal calf serum. After two passages, a confluent, homogeneous monolayer of cells with fibroblast morphology was present. These cells were positive for vimentin and negative for cytokeratin by immunocytochemistry. Adult rat lung fibroblasts were isolated from 9-wk-old rats. These cells were in approximately the 12th to 15th passages and were derived from lung tissue that had been minced and pressed through a 100-mesh screen before culture. After three passages, a confluent, homogeneous population of cells with a fibroblast morphology was obtained. These cells were vimentin positive and cytokeratin negative. Rat lung microvascular endothelial cells isolated from adult rats with the technique of Magee et al. (18) were a generous gift from Dr. Andrew Canada (Duke University Medical Center, Durham, NC). A rat lung alveolar epithelial cell line was derived from rat primary neonatal type II cells by transfection with SV40 large T antigen. These epithelial cells were cytokeratin positive and demonstrated serum-dependent proliferation. The cells were maintained in DMEM with 10% fetal calf serum. All media were supplemented with penicillin and streptomycin. Cultures were grown in humidified 5% CO2 and 95% air at 37°C.
RNA extraction and Northern blot analysis.
Total RNA was prepared from lung tissues and cells essentially with the
method of Chirgwin et al. (6) by extracting tissue with 4 M guanidinium
thiocyanate and pelleting through a cushion of cesium chloride. The
cDNAs for rat TR-I and T
R-II were isolated from rat lung as
previously described (40). The cDNAs for rat TGF-
1 (28), mouse
TGF-
2 (23), and mouse TGF-
3 (7) were obtained from the American
Type Culture Collection (Manassas, VA). Poly(A)+ RNAs were
selected with the PolyATtract mRNA isolation kit (Promega, Madison,
WI). The integrity and quantity of the RNA were evaluated by
ultraviolet spectrophotometry and denaturation of agarose gels stained
with ethidium bromide. Two micrograms of mRNA were fractionated on an
agarose gel, transferred to Biotrans nylon membrane (ICN, Irvine, CA),
and fixed in a Stratalinker UV cross-linker (Stratagene, La Jolla, CA).
Filters were hybridized at 42°C in 50% formamide solution
containing 5× saline-sodium phosphate-EDTA (SSPE; 1× SSPE
is 0.18 M NaCl, 10 mM Na2HPO4, and 1 mM EDTA),
5× Denhardt's solution [5× Denhardt's is 0.1%
(wt/vol) each of polyvinylpyrrolidone, bovine serum albumin, and
Ficoll], 0.1% SDS, and 0.1 mg/ml of denatured and sonicated
salmon sperm DNA with 106 counts/min of
32P-labeled T
R-I, T
R-II, TGF-
1, TGF-
2, or
TGF-
3 probe. A glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA
probe was used as a control. Filters were washed twice with 2×
saline-sodium citrate (SSC; 1× SSC is 0.15 M NaCl and 15 mM
trisodium citrate) containing 0.1% SDS for 15 min at room temperature
and finally washed with 0.1× SSC containing 0.1% SDS for 20 min
at 60°C. The filters were autoradiographed.
Reverse transcription-polymerase chain reaction. We isolated lungs from 25 gestational day 14 rat fetuses. Reverse transcription-polymerase chain reaction (RT-PCR) assay was performed as described earlier (38). Briefly, cDNA was synthesized from total RNA primed with oligo(dT)12-18 and reverse transcribed in a final volume of 20 µl with Superscript II reverse transcriptase (GIBCO BRL, Life Technologies, Grand Island, NY). The target sequences were amplified by PCR in a final volume of 50 µl. PCR cycling conditions were 28 cycles consisting of 94°C for 1 min, 55°C for 1 min, and 72°C for 1 min, followed by a 5-min final extension at 72°C. The PCR product was analyzed by agarose gel electrophoresis.
The primers for TIn situ hybridization.
Sections of paraformaldehyde-fixed and paraffin-embedded rat lungs were
subjected to in situ hybridization with 35S-cRNA. Sense and
antisense cRNA riboprobes were generated from rat TR-I cDNA
subcloned into pGEM-7Zf (Promega). The plasmid was linearized with
restriction enzymes. cRNA was obtained in the sense and antisense
orientation by transcribing from T7 and SP6 promoters, respectively,
with the riboprobe transcription kit (Stratagene). Riboprobes were
labeled with 35S-UTP (Amersham, Arlington, IL). Primary
transcripts were reduced to an average fragment length of 200 bp by
alkaline hydrolysis. Sections were rehydrated, fixed in 4%
paraformaldehyde in PBS, and treated with proteinase K. The sections
were treated again with 4% paraformaldehyde in PBS and acetylated with
acetic anhydride. After dehydration through ethanol, the sections were
prehybridized in hybridization solution containing 50% formamide, 0.3 M NaCl, 20 mM Tris · HCl, pH 8.0, 5 mM EDTA, 0.02%
polyvinylpyrrolidone, 0.02% Ficoll, 0.02% bovine serum albumin, 500 µg/ml of tRNA, and 100 mM dithiothreitol. Hybridization was performed
with 2-5 × 107
counts · min
1 · ml
1
of 35S-labeled cRNA probe in hybridization solution (with
10% dextran sulfate) and incubated overnight at 55°C. The slides
were washed with 5× SSC containing 10 mM dithiothreitol at
37°C, followed by washing with 2× SSC at 65°C, and then
treated with RNase A. Washing was then continued with 2× SSC at
65°C and 0.1× SSC at 37°C. Sections were next dehydrated,
dried, and dipped in Kodak NTB-2 autoradiographic emulsion. After
exposure at 4°C for 1-2 wk, the slides were developed and
counterstained with hematoxylin and eosin.
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RESULTS |
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Detection of TR-I mRNA in developing rat lung by
Northern blot analysis.
Developmental expression of T
R-I was studied in rat lungs. We
isolated RNA from embryonic rat lungs of day 14 and day
16 gestational ages and performed RT-PCR analysis. We detected both T
R-I and T
R-II expression in gestational day 14 and
day 16 rat lungs, and the level of T
R-I and T
R-II
expression in gestational day 14 rat lung was slightly lower
than that in day 16 rat lung (Fig.
1). We analyzed T
R-I mRNA levels in rat
lungs at different embryonic and postnatal stages by Northern blotting
(Fig. 2). A 6.1-kb T
R-I transcript was
detected in rat lung tissue. The level of expression of T
R-I mRNA
changed significantly during lung development. T
R-I mRNA was
detected in rat fetal lung tissues early in development, reached
maximal concentration at 16 days of gestation, and then decreased as
development proceeded. T
R-II showed a different expression pattern
from that of T
R-I over the same developmental times. T
R-II mRNA
was increased as development progressed, reached maximal concentration
in the early postnatal period, and then decreased.
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Localization of TR-I in fetal and postnatal rat lung
tissues by in situ hybridization.
The spatial localization of T
R-I in fetal and postnatal rat lung
tissues was investigated by using in situ hybridization performed with
antisense cRNA probes. Figure 4 shows in
situ hybridization of T
R-I mRNA in a gestational day 14 rat
embryo. T
R-I mRNA was observed in the mesenchyme and epithelium of
gestational day 14 rat lungs. The most prominent labeling
signal of T
R-I was noted in the developing epithelium.
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Expression of TR-I by cultured lung cells.
To investigate the cell-type expression of T
R-I in vitro, we
examined the expression of T
R-I by four lung-derived stable cell
lines (Fig. 8). T
R-I mRNA was detected
as a 6.1-kb species expressed in adult rat lung microvascular
endothelial cells, alveolar epithelial cells, fetal lung fibroblasts,
and adult lung fibroblasts. Two T
R-I mRNA species of ~6.1 and 4.0 kb were observed in lung microvascular endothelial cells and fetal lung
fibroblasts.
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DISCUSSION |
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The mechanism of lung development involves epithelial-mesenchymal
interactions, which are spatially and temporally controlled. TGF- is
a family of multifunctional cytokines that are involved in lung
development. Overexpression of TGF-
1 in the developing respiratory
epithelium of transgenic mice inhibits lung sacculation and epithelial
cell differentiation, indicating a role for TGF-
1 in lung
morphogenesis and differentiation (42). TGF-
2 knockout mice show
postnatal defects in the conducting airways of the lung but have no
gross morphological defects in prenatal lungs (30). The TGF-
3
knockout, in contrast to the TGF-
1 and TGF-
2 knockouts, produced
a profound inhibition of lung development and was lethal at birth due
to respiratory insufficiency (13). T
R-I and T
R-II have been
identified as the signaling receptors for TGF-
. Zhao and Young (40)
previously demonstrated that the expression of T
R-II is spatially
and temporally regulated during rat lung development. To gain further
clues about the function and mechanism of TGF-
signaling during lung
development, we examined the temporal and spatial expression of T
R-I
during lung development.
The expression of TR-I mRNA was found in mesenchymal tissues and
epithelium at all stages of lung development, which overlapped the
expression of T
R-II (40). The finding that T
R-I is ubiquitously expressed in the lung was anticipated because TGF-
has been found to
act on many different cell types and to regulate a wide variety of
cellular activities in the lung (26), and both T
R-I and T
R-II are
directly involved in receptor signal transduction (4, 36). Expression
of a truncated T
R-II lacking the intracellular kinase domain
suppresses cell growth signal by TGF-
in Mv1Lu mink lung epithelial
cells (4, 35) and in lung fibroblasts (41). Both T
R-I and T
R-II
have been shown to be required for the regulation of extracellular
matrix production in rat lung fibroblasts by TGF-
(39).
The expression of TR-I by four lung-derived stable cell lines was
examined. T
R-I mRNA was expressed in adult rat lung microvascular endothelial cells, alveolar epithelial cells, fetal lung fibroblasts, and adult lung fibroblasts. Numerous in vitro studies show that TGF-
elicits a multiplicity of effects in various lung-derived cell types
(reviewed in Ref. 26). The expression of TGF-
receptors by these lung-derived cell types, together with those previously reported in vitro studies of TGF-
biological effects on
lung-derived cell types, suggests that TGF-
s and TGF-
receptors
might be needed for the ongoing regulation of growth in the normal
function of mature lung cells beyond the fetal period.
TGF- was initially named by its ability to promote
anchorage-independent growth and a transformed phenotype in
nontumorigenic mesenchymal cells (24, 29). The localization of T
R-I
in mesenchymal cells of the developing lung supports a role for TGF-
signaling in regulating the growth of embryonic lung connective tissues during development. In addition to mediating signals controlling the
growth of lung connective tissue, T
R-I is also involved in TGF-
signaling that regulates the synthesis of extracellular matrix
components in lung fibroblasts (39, 41). It is clear that the
mesenchyme controls the airway branching pattern and that processes
inherent in the epithelial cells alter cell and tissue shape to produce
branch points (32). Cell-extracellular matrix interactions are known as
one class of important mechanisms that direct development of structural
and functional lungs (10, 22). Regulation of lung fibroblast
extracellular matrix molecule biosynthesis through TGF-
-receptor
signal transduction could pass signals from the mesenchyme to the
epithelium, thus governing a cascade of reciprocal interactions between
the mesenchyme and epithelium and regulating the ensuing morphogenesis
of the forming lung.
We found that a high level of TR-I mRNA was expressed in rat fetal
lung tissue early in development, but the expression of T
R-I mRNA
decreased as development proceeded. The temporal expression of T
R-I
was different from that of T
R-II during rat lung development. The
expression of T
R-II mRNA was low in rat fetal lung tissue early in
development, but the expression of T
R-II mRNA increased as
development continued. TGF-
receptors and their ligands exhibit distinct but overlapping patterns of expression during lung
development. The time course of T
R-I expression was similar to those
of TGF-
2 and TGF-
3 at early developmental stages, and their
expression levels were highest in rat fetal lung tissue early in
development. However, the expression of T
R-I was different from that
of TGF-
2 and TGF-
3 during the postnatal period. A similar
temporal expression was observed for T
R-II and TGF-
1, and their
expression levels were maximal in postnatal day 1 rat lung. The
apparent differential expression of the three TGF-
isoforms and
their receptors at different developmental stages raises interesting
possibilities of specific roles for each ligand and for T
R-I and
T
R-II during lung development in vivo.
The finding that TR-I and T
R-II were differentially expressed in
the process of lung development was not anticipated. A ligand-induced
heterooligomer model was proposed for TGF-
signal transduction (33,
36, 37), in which the biological effects of TGF-
are mediated by
T
R-II in concert with T
R-I. TGF-
binds directly to T
R-II,
which then recruits T
R-I to form a heteromeric complex. The
signaling TGF-
-receptor complex contains two molecules each of
T
R-I and T
R-II bound to one TGF-
molecule. Our results show
that the expression level of T
R-I was distinct from that of T
R-II
during rat lung development, which indicates that an excessive amount
of either T
R-I or T
R-II may exist. The individual roles of
excessive T
R-I and T
R-II remain to be elucidated. The ratio of
T
R-I to T
R-II may influence the biological activities of TGF-
.
The expression of TR-I and T
R-II is regulated distinctly not only
in a temporal manner but also in a spatial manner during lung
development. A previous study by Zhao and Young (40) showed an
interesting expression pattern of T
R-II in day 16 gestational age rat fetal lung tissue. T
R-II was expressed along a
proximal-distal gradient in the developing airway epithelium. A
concentrated expression of T
R-II was detected in the distal cuboidal
epithelium, whereas the T
R-II transcript was absent or in low
concentration in the proximal pseudostratified columnar epithelium. In
contrast, we found that the epithelial cells in both the distal and
proximal bronchioles had a similar level of T
R-I expression. TGF-
has been recognized as being important in lung epithelial cell
differentiation (12, 27). Based on these observations, we speculate
that both T
R-I and T
R-II may participate in the TGF-
signaling
pathway to regulate lung epithelial cell differentiation, but T
R-II
may provide positional information during epithelial morphogenesis.
In conclusion, we described the expression and localization of TR-I
mRNA during lung development. The expression of T
R-I was found to be
highly regulated during lung development. Compared with T
R-II,
T
R-I shows a unique developmental expression and localization
pattern in rat lung. The temporal and spatial distribution of T
R-I
mRNA in rat lung tissues along with its regulated expression pattern
during development supports the notion that TGF-
plays an important
role as a multifunctional autocrine/paracrine regulator during lung
development. The distinction of the temporal and spatial expression of
T
R-I and T
R-II mRNAs in the rat lung suggests that each receptor
may play a different role in lung development. To ascertain their roles
in the signal transduction pathway and the diverse regulatory effects
of TGF-
in lung development processes, further experiments remain to
be done.
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ACKNOWLEDGEMENTS |
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This work was supported by a Department of Veterans Affairs grant; National Heart, Lung, and Blood Institute Grant HL-32188; and an American Lung Association grant.
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FOOTNOTES |
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Y. Zhao is a recipient of the Clifford W. Perry Research Award from the American Lung Association of North Carolina.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: Y. Zhao, Medical Research 151, Durham VA Medical Center, Durham, NC 27705 (E-mail: zhaoyun{at}duke.edu).
Received 7 October 1999; accepted in final form 11 January 2000.
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