(Received for publication, October 11, 1995; and in revised form, December 12, 1995)
From the
Growth regulation of fibroblasts is important for lung
development and repair of lung injury. In this study, we investigated
the role of transforming growth factor- (TGF-
) type II
receptor in the TGF-
-dependent proliferative response of lung
fibroblasts. TGF-
stimulated the proliferation of adult lung
fibroblasts at a low concentration (1 ng/ml), but inhibited the growth
of fetal lung fibroblasts in a dose-dependent fashion (0.1-10
ng/ml). Cross-linking and Northern analysis demonstrated that the two
lung fibroblast cell lines expressed the TGF-
type I receptor
(T
RI) and type II receptor (T
RII). We overexpressed in lung
fibroblasts a truncated derivative of T
RII that lacked the
cytoplasmic serine/threonine kinase domain (T
RII
K).
T
RII
K was a dominant-negative inhibitor of TGF-
signal
transduction blocking not only TGF-
-induced mitogenic action upon
adult lung fibroblasts but also TGF-
-induced growth inhibition of
fetal lung fibroblasts. The results indicate that the type II receptor
is indispensable for mediating both the mitogenic and antiproliferative
effects of TGF-
upon lung fibroblasts.
Transforming growth factor- (TGF-
) (
)is a
family of multifunctional cytokines that regulates cell growth,
differentiation, and extracellular matrix deposition(1) . Three
isoforms TGF-
1, -2, and -3 have been identified in lung and found
to act on many different lung-derived cell types and to regulate a wide
variety of cellular activities(2, 3) . TGF-
s
elicit their biological effects on cells through binding to cell
surface transmembrane receptors. A number of different types of
putative receptors for TGF-
, including three distinct size classes
termed type I (T
RI, 50-60 kDa), type II (T
RII,
75-85 kDa), and type III (T
RIII, a 280-kDa proteoglycan with
a 120-kDa core protein), have been identified by affinity cross-linking
experiments(4, 5) . Molecular cloning of cDNAs coding
type I and type II receptors for the TGF-
superfamily have shown
that both types belong to a novel family of transmembrane
serine/threonine kinases with a small extracellular domain, a single
transmembrane segment, and an intracellular region with a
serine/threonine kinase domain(6, 7, 8) .
Sequence analysis of T
RIII revealed that it is a transmembrane
proteoglycan with a short and highly conserved cytoplasmic domain that
has no apparent signal motif (9, 10) . Current
evidence indicates that a complex of T
RI and T
RII, but not
the individual components, mediates TGF-
signal transduction. A
ligand-induced heterodimer model was proposed for TGF-
signal
transduction(11, 12) .
TGF- may act as either
a positive or a negative regulator of cell division. TGF-
stimulates the proliferation of mesenchyme-derived cells such as
fibroblasts and osteoblasts, but acts as a powerful growth inhibitor of
cells of epithelial and endothelial origin(13, 14) .
TGF-
inhibits epithelial cell proliferation by delaying or
arresting progression through the late portion of G1(15) .
T
RII was found to be essential for TGF-
growth inhibition
signal. Mv1Lu mink lung epithelial cells are highly responsive to the
growth inhibition of TGF-
. A chemically mutated Mv1Lu cell line
defective in T
RII lacks TGF-
-induced growth inhibition.
Transfection of the human T
RII to this mutant cell line restored
the inhibition of growth by TGF-
(16, 17) . A
similar conclusion has been drawn by expression of a kinase-defective
truncation of the human T
RII in Mv1Lu mink lung epithelial
cells(18) .
TRII is required to mediate
antiproliferative responses to TGF-
, but its involvement in
TGF-
signaling that lead to growth stimulation has not been
established. In the present studies, we demonstrate a bifunctional
action of TGF-
in lung fibroblast cells. An adult rat lung
fibroblast cell line expressed T
RII and was responsive to the
growth stimulation of TGF-
, whereas a fetal rat lung fibroblast
cell line expressed T
RII and displayed an unexpected response,
growth inhibition, to TGF-
. To further determine the role of
T
RII in modulating the growth stimulation effects of TGF-
, we
transfected into rat lung fibroblast cells an expression plasmid
containing rat T
RII cDNA that was lacking the kinase domain.
Overexpression of the dominant-negative T
RII mutant blocked both
the stimulating and the inhibitory effects of TGF-
on rat lung
fibroblasts. These experiments provide evidence for a functional role
of T
RII in TGF-
-induced proliferation and growth inhibition
of lung fibroblasts.
Growth responses to TGF-1 by rat lung fibroblasts were
examined. Confluent fetal rat lung fibroblasts and adult rat lung
fibroblasts were made quiescent and exposed to different concentration
of TGF-
1. The adult rat lung fibroblast cell line was responsive
to the growth stimulation of TGF-
as expected of most fibroblast
cells, whereas fetal rat lung fibroblasts displayed an unexpected
response, growth inhibition (Fig. 1). The effects of TGF-
1
on the proliferation of adult lung fibroblasts were dependent on
concentration (Fig. 1A). TGF-
1 stimulated the
proliferation of adult lung fibroblasts at concentration less than 5
ng/ml. Maximal effects were observed at a concentration of 1 ng/ml, and
TGF-
1 had no effect at concentrations of over 5 ng/ml. In
contrast, TGF-
1 inhibited the proliferation of fetal lung
fibroblasts in a dose-dependent manner up to 10 ng/ml after a 20-h
treatment with TGF-
1 (Fig. 1B).
[
H]Thymidine incorporation peaked at 24-36
h in adult lung fibroblasts treated with TGF-
1, but inhibition of
DNA synthesis of fetal lung fibroblasts by TGF-
1 persisted for up
to 72 h (data not shown).
Figure 1:
Effects of TGF-1 on growth of
adult lung fibroblast cells (A) and fetal lung fibroblasts (B). Lung fibroblasts were plated at a density of 2
10
cells/well in a 24-well plate and were made quiescent in
serum-free DMEM for 24 h. Cells were treated with the indicated
concentration of TGF-
1 for 20 h and then labeled with
[
H]thymidine for 6 h. DNA was precipitated with
10% trichloroacetic acid, and the amount of
[
H]thymidine incorporated was analyzed by liquid
scintillation counting. Each value represents the average of three
separate cultures measured for each TGF-
1 concentration used. Error bars are S.E.
In order to elucidate a possible mechanism
whereby TGF-1 exerts its actions on fibroblast proliferation, the
expression of TGF-
receptors by adult and fetal lung fibroblasts
were evaluated. Cross-linking of
I-TGF-
1 to fetal
lung fibroblasts or adult lung fibroblasts revealed three species of
receptors with apparent molecular masses of 60, 85 and 280 kDa (Fig. 2A). These proteins were equivalent to the
TGF-
type I, II, and III receptors, respectively. A 40-kDa
component was seen in fetal lung fibroblasts, but not in adult lung
fibroblasts. It is not clear whether it was an isoform of the TGF-
type I receptor or an uncharacterized TGF-
binding protein. A
lower molecular mass species (35 kDa) was also detected in both adult
and fetal lung fibroblasts.
Figure 2:
Expression of endogenous TGF-
receptors in lung fibroblasts. A, receptor cross-linking
assays were used to measure cell surface TGF-
receptor expression.
Monolayer cultures of adult lung fibroblast cells (lane 1) or
fetal rat lung fibroblasts (lane 2) in six-well plates were
incubated with 100 pM of
I-TGF-
1. The
receptors were cross-linked with disuccinimidyl suberate. Cell extracts
were resolved on a SDS-polyacrylamide gel under reducing conditions.
The TGF-
receptor type III (T
RIII), type II (T
RII), and type I (T
RI) were visualized
after autoradiography; B, Northern blotting was used to
analyze expression of T
RI and T
RII mRNAs. Each lane contained
2 µg of mRNA prepared from adult lung fibroblasts (lane 1)
or fetal lung fibroblasts (lane 2). mRNA was subjected to
electrophoresis through a formaldehyde denaturing agarose gel and,
after Northern blotting, hybridized with radiolabeled T
RI or
T
RII cDNA probe and quantified by scanning densitometry. Scan
values for T
RI and T
RII mRNA signals were normalized with the
scan data of glyceraldehyde phosphate dehydrogenase. Ratios were
expressed in arbitrary units.
The expression of TGF- type I and
II receptor were further examined by Northern blot analysis (Fig. 2B). T
RII mRNA was detected as a
5.1-kilobase species expressed in adult lung fibroblasts and in fetal
lung fibroblasts. Two T
RI mRNA species of approximately 6.1 and
4.0 kilobases were observed in adult lung fibroblasts and in fetal lung
fibroblasts. T
RII and T
RI were differentially expressed in
fetal and adult lung fibroblasts.
To examine the significance of
type II receptor in mediating the growth-promoting and growth
inhibition effects of TGF-, we used a dominant-negative inhibitory
approach to create a loss-of-function mutation of T
RII. We
constructed a kinase-deficient cytoplasmic deletion mutant of rat
T
RII (T
RII
K). The truncated T
RII and wild type
T
RII were cloned into pcDNA3, under the transcriptional control of
the cytomegalovirus immediate early gene promoter and enhancer. We
transfected rat lung fibroblasts with rat T
RII
K or T
RII.
The expression level of the truncated receptor was tested by affinity
labeling of transfected cells with
I-TGF-
1 (Fig. 3). Fibroblasts transfected with T
RII
K yielded a
TGF-
affinity-labeled product of 45 kDa, the predicted size of the
truncated receptor T
RII. A high level of T
RII
K were
expressed on the cell surface of fetal and adult lung fibroblasts. The
truncated receptor was able to bind ligand.
I-TGF-
1
binding to T
RII
K was efficiently competed by unlabeled
ligand, the same as the wild type T
RII binding.
Figure 3:
Expression of the truncated TRII
lacking the cytoplasmic kinase domain in lung fibroblasts. Monolayer
cultures of fetal rat lung fibroblasts or adult rat lung fibroblasts
transiently transfected with rat T
RII
K or empty pCDNA3 vector
were incubated with
I-TGF-
1 in the absence(-)
or in the presence (+) of 80 nM unlabeled TGF-
1.
Bound
I-TGF-
1 was cross-linked with disuccinimidyl
suberate. Cell lysates were subjected to SDS-polyacrylamide gel
electrophoresis under reducing conditions and then were
autoradiographed. Lanes 1, 2, 5, and 6, fetal lung
fibroblasts; lanes 3, 4, 7, and 8, adult lung
fibroblasts.
[H]-thymidine incorporation was measured to
assess the proliferation rate of lung fibroblasts expressing the
truncated T
RII. Adult lung fibroblasts were transfected with
T
RII
K or empty vector. Transfected fibroblasts were cultured
in the absence or presence of TGF-
1 (1 ng/ml) for 20 h.
T
RII
K completely blocked the TGF-
1-dependent DNA
synthesis (Fig. 4A). When adult lung fibroblasts were
transfected with 1 µg of T
RII
K and 1 µg of wild type
rat T
RII, the growth responsiveness of adult lung fibroblasts to
TGF-
1 was restored by adding the wild type T
RII cDNA (Fig. 4B). Fetal lung fibroblasts transfected with
T
RII
K were unable to convey TGF-
1-dependent growth
inhibition (Fig. 5A). Similarly, the diminished
response to TGF-
1 by fetal fibroblasts was rescued by
cotransfection of T
RII
K with the wild type T
RII (Fig. 5B). The results show that the kinase-defective
deletion of T
RII is a dominant-acting inhibitor of signal
transduction by TGF-
receptor complex and that T
RII was
required for TGF-
-dependent positive and negative growth response.
Figure 4:
Dominant-negative effects of TRII on
TGF-
-induced growth. A, adult lung fibroblasts were
transfected with T
RII
K or empty vector. Transfected
fibroblasts were cultured in the absence or presence of TGF-
1 (1
ng/ml) for 20 h. DNA synthesis was assayed by measuring
[
H]thymidine incorporation; B,
restoration of TGF-
1-dependent growth response. Adult lung
fibroblasts were transfected with 1 µg of T
RII
K and 1
µg of wild type rat T
RII. The cells were treated with
TGF-
1 for 20 h and then assayed for DNA synthesis. The bars represent the means ± S.E. (n =
3).
Figure 5:
Dominant-negative effects of TRII on
TGF-
-induced antiproliferative response. A, fetal lung
fibroblasts were transfected with T
RII
K or empty vector.
Transfected fibroblasts were cultured in the absence or presence of
TGF-
1 (5 ng/ml) for 20 h. DNA synthesis was assayed by measuring
[
H]thymidine incorporation; B,
restoration of TGF-
1-dependent growth inhibition. Fetal lung
fibroblasts were cotransfected with 1 µg of T
RII
K and 1
µg of wild type rat T
RII. The cells were treated with
TGF-
1 for 20 h and then assayed for DNA synthesis. The bars represent the means ± S.E. (n =
3).
Proliferation of fibroblasts is an important aspect of lung
development and is also a key feature of repair of lung injury.
Expression of TGF- isoforms has been detected in the lung at
critical times during development(2, 23) . The
expression pattern and known biological activities of TGF-
suggest
an important role for this factor in lung development. In this study,
the effects of TGF-
on growth of fetal and adult lung fibroblasts
were characterized, and the role of T
RII in TGF-
-induced
signaling was examined.
Our data show that TGF- stimulates
proliferation of adult fibroblasts at low concentration but remains
inactive at higher concentration, while inhibiting growth of fetal
fibroblasts regardless of concentration. An example of the bifunctional
nature of TGF-
has been reported in human smooth muscle
cells(24) . TGF-
is mitogenic only when used at lower
concentration, whereas at higher concentration it inhibits DNA
synthesis. More recent studies have demonstrated that TGF-
can act
as a negative or positive growth regulator in two sublines of the same
epithelial cells, depending on their commitment to
differentiation(25) . TGF-
activates two different signal
transduction pathways through activating different Ras proteins and
myelin basic protein kinases(26) .
We examined the possible
relationship between the bifunctional action of TGF- and the
expression of its cellular receptors. The two fibroblast cell lines
possess all three TGF-
receptors; however, fetal fibroblasts
expressed a much higher level of cell surface type II receptor at the
mRNA and the protein level than adult lung fibroblasts. The finding is
in agreement with our previous study (20) that the expression
of T
RII is developmentally regulated in the lung. This raises the
possibility that the bifunctional action of TGF-
on lung
fibroblasts may depend on their developmental stage.
We show that
the kinase-deleted truncation of TRII acts as a dominant inhibitor
of TGF-
signal transduction. The proliferative action and the
growth inhibition of TGF-
were abolished by overexpression of
T
RII
K. TGF-
-induced signaling events leading to growth
stimulation are likely to be different at some level from that of
growth inhibition. TGF-
increases c-fos expression in
growth-inhibited cells but not in growth-stimulated cells (27) . An increase in retinoblastoma protein phosphorylation is
seen in TGF-
-induced proliferation(28) . However, our
results indicated T
RII is essential for both pathways. This
finding suggests that binding of TGF-
to T
RII may be a common
step required for the TGF-
signaling cascade. The TGF-
signal
transduction pathways for the growth stimulation and the growth
inhibition may diverge after binding of TGF-
to T
RII and then
different intracellular signals might be activated. An intriguing
question raised by this study is how TGF-
exerts its multiplicity
of effects through interaction with its transmembrane receptors. The
signaling specificity could occur through interaction of T
RII with
different types of type I receptor. The characterization of other
members of TGF-
receptor family, particularly type I receptors,
might allow us to determine the mechanism of multiple effects of
TGF-
.
In conclusion, we found that TGF- stimulates
proliferation of adult lung fibroblasts, while inhibiting growth of
fetal lung fibroblasts. We have shown that the truncated form of
T
RII blocked both growth-stimulating and inhibition effects of
TGF-
. The results suggest that TGF-
type II receptor is
indispensable for the signal transduction pathway and the diverse
regulatory effects of TGF-
.