From the
Bovine aortic endothelial cells (BAECs) express both type I and
type II receptors for transforming growth factor
Transforming growth factor
The function
of the type III receptor is unclear. The TGF
Although most of the biological activities are
shared among TGF
Our laboratory has spent several
years characterizing the TGF
BAECs endogenously express both type I and type II receptors
for TGF
The expression of the type III receptor protein
increases the responsiveness of BAECs to TGF
Large vessel endothelial cells such
as BAECs and small vessel endothelial cells such as rat fat pad
capillary endothelial cells differ in the expression of the TGF
We thank Dr. Anne Romanic, Adeline Tucker, and Joanne
Lum for technical assistance. We also thank Dr. Lucia Languino for
critically reading this manuscript.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(TGF
).
These cells respond to TGF
1 but are relatively refractory to
another isoform of TGF
, termed TGF
2. TGF
s are thought to
signal through receptor complexes composed of type I and/or type II
receptors, both of which appear to be functional serine-threonine
kinases. The TGF
type III receptor, on the other hand, does not
seem to have any direct signaling capacity. We have now stably
transfected BAECs with the type III receptor cDNA. These cells
displayed surface expression of the type III receptor protein, as
determined by cross-linking with iodinated TGF
1 and
immunoprecipitation with antibodies to the type III receptor protein.
Transfected BAECs exhibit increased responsiveness to TGF
2 by
several different criteria including an increase in plasminogen
activator inhibitor-1 protein and inhibition of migration and
proliferation. Thus, the type III receptor protein may play a role in
presenting TGF
2 to the type II receptor and increase
responsiveness to TGF
2 to a level comparable to that of TGF
1.
s (TGF
s)
(
)
are multifunctional proteins that regulate cell
proliferation, differentiation, and biosynthesis of the extracellular
matrix
(1, 2, 3) . The five known mammalian
TGF
isoforms (TGF
1,
2,
3,
1.2, and
1.3)
are homo- or heterodimers linked by a single disulfide bond. When
studied under in vitro cell culture conditions, they have
overlapping functions; however it remains unclear whether the five
isoforms exert distinct physiological effects during
differentiation
(4) . Most types of cultured cells possess three
distinct high affinity cell surface receptors for TGF
as judged by
chemical cross-linking to
I-TGF
1; types I (55 kDa),
II (80 kDa), and III (280 kDa)
(3, 5, 6) .
Cloning of the cDNA encoding both type I and type II receptors revealed
a transmembrane serine-threonine
kinase
(7, 8, 9, 10, 11) . In
many studies using chemically mutagenized mink lung epithelial cells
(12-15) and tumor cell lines that are resistant to the growth
inhibitory effects of TGF
, the results most consistently
correlated with the loss of TGF
type I receptor or the loss of
both the type I and type II receptors. These observations support the
notion that TGF
-mediated signaling might involve both the TGF
type I and type II
receptors
(15, 16, 17, 18) . Expression
of the recombinant human TGF
type II receptor in certain
mutant-resistant cell lines restores sensitivity to TGF
and
results in the appearance of cell surface type II receptor as well as
the type I receptor
(8, 19) . Recently, Wrana and
co-workers
(20) provided evidence in these cell models that type
II receptor is required to activate the type I receptor which
subsequently sends the downstream signals, although these results may
not be universal
(21, 22, 23) .
type III receptor is
the most abundant TGF
receptor subtype in most cells (16, 24).
Cloning of the cDNA encoding the type III receptor, a transmembrane
proteoglycan, revealed a short highly conserved cytoplasmic domain with
no apparent signaling motif
(25, 26, 27) .
Consequently, the type III receptor and its soluble secreted ectodomain
may act as reservoir molecules for ligand retention or presentation of
TGF
to the signaling type I and type II receptors. Consistent with
this, stable transfection of recombinant rat type III receptor in
myoblasts resulted in increase in binding of TGF
1 to the type II
receptor
(26) .
1, TGF
2, and TGF
3, there are some
differences. TGF
1 and
3 elicit their effects at about
100-fold lower protein concentration compared to TGF
2 in certain
cell types that lack type III receptor. Indeed vascular endothelial
cells and hematopoietic progenitor cell lines that lack type III
receptor are relatively resistant to TGF
2 (17, 24, 28-32).
Expression of type III receptor in L
E
myoblasts
that endogenously lack the protein results in enhanced binding of
TGF
2 to the type II receptor and suggests interactions between the
types II- and III-binding sites (33, 34). Based on the structural
features of the type III receptor and the observation that cells that
lack this receptor still respond to TGF
, the role of type III
receptor is most likely in modulating the TGF
isoform
response
(16, 28) .
responses of vascular endothelial
cells, comparing the effects of TGF
1, TGF
2, and TGF
3 on
bovine aortic endothelial cells (BAECs). TGF
1 and TGF
3
profoundly inhibit BAEC proliferation and migration, while TGF
2
has little or no effect. To test the hypothesis that the type III
receptor regulates the binding of TGF
2 to the signaling receptors
and increases the TGF
2 responsiveness in BAECs, we generated
stable transfectants of BAECs expressing the type III receptor cDNA in
both the sense and antisense orientations. We now present changes in
BAEC responsiveness to TGF
2 that are comparable to TGF
1
effects when type III receptors are present.
Materials
Human TGF1 and porcine TGF
2
were purchased from R & D Systems Inc., Minneapolis. Iodinated
TGF
1 was obtained from Biomedical Technologies Inc., Stoughton,
MA. Bovine type I collagen was isolated and purified as
described
(35, 36, 37) . Tissue culture plates
were coated with type I collagen at a concentration of 12.5 µg/ml.
Plasmids
The vector used for transfection of the
type III receptor into bovine aortic endothelial cells was obtained
from Wang and co-workers
(26) . Basically the HindIII
fragment of the rat type III receptor cDNA (R3-OFF) was subcloned into
pcDNA I NEO (Invitrogen, San Diego, CA) which is under the control of
the cytomegalovirus transcriptional promoter and the SV40 origin of
replication. In addition, two other constructs were generated by
subcloning a 3.9-kilobase EcoRI fragment from the rat type III
receptor cDNA (a gift from J. Massague, Memorial Sloan Kettering Cancer
Center, NY) into the pSV7d expression vector
(38) which is under
the control of the Rous sarcoma virus transcriptional promoter. The
orientation of the EcoRI fragment was checked by restriction
digests with AflIII and SalI. Thus the rat type III
receptor cDNA was subcloned into pSV7d in both the sense and antisense
orientations. A control vector pcDNA3 (Invitrogen, San Diego, CA)
containing the neomycin resistance marker was cotransfected into the
cells along with the type III receptor cDNA subcloned into the pSV7d.
Cell Culture and Transfection
Bovine aortic
endothelial cells were isolated and cultured according to Madri and
Furthmayr
(36) . For the transfection experiments, cells were
plated on 60-mm dishes at subconfluence. The cells were transfected
with 10 µg of plasmid DNA and 25 µg of lipofectAMINE
(Life Technologies, Inc.) per plate and incubated with serum-free media
(Life Technologies, Inc.) for several hours before growth media was
added. Selection for transfectants with 400 µg/ml G418 (Geneticin,
Life Technologies, Inc.) was carried out 48 h after the transfection.
Neomycin -resistant colonies were selected using cloning rings and
expanded before analysis for surface expression of the receptors.
Receptor Binding Autoradiography Assay
The assay
was a modification of our earlier studies
(32, 39) .
Briefly, confluent monolayers grown on 35 mm, 1.5% gelatin-coated
tissue culture plates were washed with cold binding buffer
(Dulbecco's modified essential media, 25 mM HEPES, pH
7.4, 0.1% bovine serum albumin) and then allowed to equilibrate with
binding buffer for 30 min at 4 °C on a rotating platform. The
buffer was aspirated, and 250 µl of ice-cold binding buffer
containing 100 pMI-labeled TGF
1 (220
µCi/µg) was added to each 35-mm dish and incubated on a rotary
platform at 4 °C for 3 h. After washing at 4 °C, ice-cold
binding buffer lacking bovine serum albumin was added to the plates. 5
µl of 27 mM disuccinimidyl suberate (Pierce) was added to
each plate. The plates were swirled immediately after addition of the
disuccinimidyl suberate. The dishes were allowed to shake on the rotary
platform for 15 min. The medium was removed and washed several times
with binding buffer before lysis with Laemmli loading buffer containing
30 µl of 1 mM dithiothreitol. The cell lysate was boiled
and electrophoresed according to Laemmli
(40) . Linear gradient
resolving gels of 5-10% polyacrylamide were constructed with a
3.5% stacking gel. The gels were fixed, dried, and exposed to Amersham
Hyperfilm
-MP at -80 °C. The films were developed
using a Kodak X-OMAT M20 processor (Kodak).
Surface Biotinylation
Cells plated on 100-mm
dishes were washed with PBS containing 0.2 mM calcium chloride
and 2 mM magnesium chloride and incubated for 30 min at 4
°C with 1 mg of NHS-Long Chain Biotin (Pierce) in 2 ml of PBS
containing calcium chloride and magnesium chloride. The cells were
washed with PBS and lysed with 0.05% Triton X-100, 120 mM
Tris-HCl, pH 8.7. The cell lysates were spun at 14,000 revolutions/min
in an Eppendorf centrifuge for 10 min at 4 °C, and the supernatant
was used for immunoprecipitation.
Immunoprecipitation
Cell lysates obtained from
I-TGF
1 cross-linking experiments and surface
biotinylation experiments were incubated with primary antibodies
(anti-type III receptor and anti-type II receptor; gifts from F.
Lopez-Casillas, Memorial Sloan-Kettering Cancer Center, New York and H.
Y. Lin, Whitehead Institute for Biomedical Research, Cambridge, MA,
respectively) overnight on a rotary shaker. 50 µl of protein-A
Sepharose CL 4B (Pharmacia) was added to the cell lysate and further
incubated for 2 h before spinning the beads down and washing the
Sepharose beads with Tris-buffered saline containing 0.5% Triton X-100
(TBS-T) several times. The beads were resuspended in sample loading
buffer and boiled for several minutes before loading onto the gel.
Immunoblot Analysis
Cell protein was extracted
with 0.05% Triton X-100 and 120 mM Tris-HCl, pH 8.7,
normalized for total protein using the bicinchonic acid assay (Pierce),
and electrophoresed through a 10% non-reducing polyacrylamide gel.
Proteins were transferred to a nitrocellulose membrane (Schleicher and
Schuell), blocked with 8% nonfat milk in PBS, and incubated with mouse
anti-bovine PAI-1 IgG (American Diagnostica Inc.) at 1:750 dilution and
then goat anti-mouse secondary antibody conjugated to horseradish
peroxidase (Promega, Madison, WI) at 1:10 000 dilution. The blot was
developed using the Enhanced Chemiluminescence method (ECL, Amersham
Corp.) with Hyperfilm-MP. Bands were quantitated using a
Molecular Dynamics densitometer (Molecular Dynamics Scanner, Sunnyvale,
CA).
Reverse Zymography
Cell extracts were normalized
for cell protein using the bicinchonic acid assay and then
electropheresed in a 10% acrylamide gel. The gel was then washed in
2.5% Triton X-100 for 1 h followed by two 20-min washes in water. The
gel was then overlaid on a thin agar gel with final concentrations of
4% nonfat milk, 0.1 M Tris-,HCl pH 8.0, 8 µg/ml
plasminogen, 1.25% agar. The gel and overlay were incubated at 37
°C until the generated plasmin had diffused throughout the entire
gel, and all the casein was degraded except where PAI-1 was located.
The gels were then examined and photographed with darkfield
illumination.
Proliferation Assays
Collagen I-coated
bacteriological cultures dishes were washed with PBS before the
addition of cell suspension (1 10
cells/dish). The
cells were allowed to attach to the coated dishes for several hours. At
this point, fresh medium with or without TGF
was added to the
cultures. The medium and factors were replaced once again on the third
day. Cell numbers were determined by lifting the cells off the culture
dishes with trypsin/EDTA and counting quadruplicate samples using a
Coulter Counter (Coulter Electronics Inc, Hialech, FL). The mean number
of cells/dish was then calculated.
Migration Assay
Cell migration was evaluated as
described previously
(41, 42) . Briefly, after cells
plated within fences located in the center of the cultures achieved
confluence (about 6 h), migration was induced by the removal of the
fences and the cells migrated outward in a radial fashion. After 6
days, the cultures were washed with PBS and fixed with 10% neutral
buffered formalin. Cultures were stained with Ham's hematoxylin.
Net increase in surface area covered was assessed using the NIH Image
Program. Maximum response was obtained from untreated control BAECs,
and this was defined as 100% relative migration.
Expression of TGF
In order to screen for potential clones expressing the
TGF Type III Receptor in
BAECs
type III receptor, we incubated the BAEC transfectants with
radioiodinated TGF
1, added chemical cross-linker, and resolved the
labeled receptor by polyacrylamide gel electrophoresis as in previous
studies
(32) . Fig. 1A shows two predominant
TGF
-binding proteins on the surface of the control BAECs (lane
1). BAECs transfected with pcDNA 3 containing the neomycin
resistance marker (lane 3) exhibited a similar binding
profile. By relative migration and comparison with other culture
models, these bands correspond to TGF
bound to the type I receptor
(65 kDa) and the type II receptor (85 kDa) complexes. Introduction of
the TGF
type III receptor cDNA in the forward orientation led to
the expression of type III receptor protein in the BAECs (lane
5). The TGF
type III receptor migrated as a diffuse band of
280-330 kDa. The identity of the molecules within the diffuse
band in the BAECIIIR cells was confirmed by immunoprecipitation with
antibodies to the TGF
type III receptor protein (lane 6).
Furthermore, introduction of the TGF
type III receptor cDNA in the
reverse orientation (pIIIRAS) profoundly reduced the surface expression
of both the type I and type II receptors (Fig. 1B,
lane 3).
Figure 1:
A,
chemical cross-linking of I-TGF
1 to stably
transfected BAECs and immunoprecipitation of cell lysates with
antibodies to TGF
IIIR. TGF
type III receptor cDNA was stably
transfected independently into BAECs either in the forward or reverse
orientations relative to the promoter of the vectors. Clonal cell lines
were incubated with
I-TGF
1, cross-linked, and
analyzed by SDS-polyacrylamide gel electrophoresis as described under
``Experimental Procedures.'' Lanes 1, 3,
and 5 show total lysates. Lanes 2, 4, and
6 show results from immunoprecipitation with antibodies to
type III receptor. The different TGF
receptor types are indicated
in the margin. Lanes 1 and 2, control BAECs;
lanes 3 and 4, BAECs transfected with pcDNA3;
lanes 5 and 6, BAECs transfected with type III cDNA
in the forward orientation. B, stable transfection of the
TGF
type IIIR cDNA in the reverse orientation significantly
reduces surface expression of the TGF
receptors. BAEC
transfectants were labeled with iodinated TGF
1, cross-linked, and
analyzed as in A. Lane 1, BAECs transfected with
vector alone; lane 2, BAECs transfected with the TGF
type
III cDNA in the forward orientation; lane 3, BAECs transfected
with the TGF
type III cDNA in the reverse
orientation.
Further Characterization of the Transfected TGF
Our initial immunoprecipitation studies in
Fig. 1
showed very little type II receptor protein
co-immunoprecipitated with the type III receptor protein similar to the
results reported by Moustakas and co-workers
(34) when they
precipitated type III receptor protein from Rat-1 cells. Therefore, to
verify that negligible levels of type II receptor were associated with
the type III receptor, we carried out immunoprecipitation reactions
with antibodies to the type II receptor protein also.
Immunoprecipitation of BAEC(V) cell lysates, which had been
cross-linked to iodinated TGF Type
III Receptor in BAECs
1, with antibodies to the TGF
type II receptor, co-precipitated the type I receptor (Fig. 2,
lane 2). Immunoprecipitation of these lysates with the type
IIIR antibodies did not precipitate any protein (Fig. 2, lane
3), indicating the absence of the TGF
type IIIR protein in
these cells. Interestingly, immunoprecipitation of BAECIIIR OE cell
lysates with antibodies to the type II receptor co-precipitated a
significant amount of the type III receptor protein (Fig. 2,
lane 5) as well, indicating a complex formation between the
TGF
type III and type II receptor proteins in the BAECIIIR cells.
Nonetheless, incubation with anti-type III receptor antibody still
failed to co-precipitate type II receptor complexes.
Figure 2:
Immunoprecipitation of BAEC TGFIIIR
with anti-TGF
IIIR and anti-TGF
IIR antibodies. BAEC
transfectants were labeled with
I-TGF
1,
cross-linked, and analyzed as in Fig. 1A. Lanes
1-3, BAECs transfected with pcDNA3; lanes 4, 5, and 6, BAECs
transfected with the type III receptor. Lanes 1 and 4 show binding in cell lysates before immunoprecipitation. Lanes
2 and 5 show results from immunoprecipitation with
antibodies to type II receptor. Lanes 3 and 6 show
results from immunoprecipitation with antibodies to type III
receptor.
Secondly, we
carried out immunoprecipitation reactions using cell lysates that had
been surface-biotinylated. Control BAECs, BAEC(V), and BAECIIIR OE
cells were surface biotinylated and equal amounts of cell lysate
incubated with antibodies to the type III receptor protein. The
immunoprecipitated proteins were detected using the
streptavidin-horseradish peroxidase and ECL detection system. Several
protein bands were detected only in BAECIIIR OE cells as shown in
Fig. 3
. Bands in the region of 280 kDa are likely to represent
the type III receptor, while the other bands noted in this
immunoprecipitation are likely to represent surface molecules that
co-precipitated with the type III receptor. Hence, immunoprecipitation
with both I-TGF
-labeled cell lysates and
biotinylated lysates indicated that BAECIIIR OE cells were indeed
expressing the type III receptor protein.
Figure 3:
Immunoprecipitation of BAEC TGFIIIR
with anti-TGF
RIII antibodies following surface biotinylation.
Control BAECs and BAEC transfectants were surface-biotinylated.
Detergent extracts were then immunoprecipitated with anti-rat
TGF
RIII antibody and resolved by 6% SDS-polyacrylamide gel
electrophoresis, transferred to a nitrocellulose membrane, and detected
using the streptavidin-horseradish peroxidase and enhanced
chemiluminescence detection system. The first lane represents
the surface-biotinylated cell lysate from control BAECs
(Lysate) whereas the rest of the lanes represent
immunoprecipitation of the respective cell lysates (Control = control BAEC; Vector = BAEC transfected
with vector alone; IIIR OE = BAEC transfected with the
TGF
type III receptor cDNA and over-expressing the TGF
type
III receptor) with antibodies to the type III receptor protein. The
molecular mass standards (194, 116, and 85 kDa) are indicated in the
margin by the arrows.
BAECs Respond to TGF
The effects of varying doses (0.5, 5.0, and 25 ng/ml)
of TGF1 by Increased PAI-1 Protein
Production and Enzyme Activity but Are Refractory to
TGF
2
1 and TGF
2 on PAI-1 protein levels and enzyme activity
in BAECs were examined by immunoblotting and reverse zymography. Both
PAI-1 protein and activity were differentially regulated by TGF
1
and TGF
2. As illustrated by the immunoblotting technique in
Fig. 4A, these amounts of TGF
1 dose-dependently
increased steady state PAI-1 protein levels while TGF
2 was
ineffective. These results were confirmed by reverse zymography as
illustrated in Fig. 4B, that is, TGF
1 increased
steady state PAI-1 activity levels at all concentrations tested, while
TGF
2 did not.
Figure 4:
A, differential effects by TGF1 and
TGF
2 on PAI-1 in BAECs. BAEC PAI-1 protein levels were determined
by immunoblotting. Lane 1 = control; lane 2 = TGF
1 (0.5 ng/ml) treatment; lane 3 =
TGF
1 (5.0 ng/ml) treatment; lane 4 = TGF
1
(25.0 ng/ml) treatment; lane 5 = TGF
2 (0.5 ng/ml)
treatment; lane 6 = TGF
2 (5.0 ng/ml) treatment;
lane 7 = TGF
2 (25.0 ng/ml); molecular mass of the
PAI-1 = 47 kDa. B, BAEC PAI-1 activity was determined
by reverse zymography.
Expression of the TGF
The data above indicated that TGF Type III Receptor Protein
Increases the Responsiveness to TGF
2 by Increased Induction of
PAI-1
1, but not
TGF
2, could induce PAI-1 protein in BAECs. We then re-examined
this issue in BAECs that expressed transfected type III receptor.
Cultures transfected with vector plasmid alone responded to TGF
1
and TGF
2 in a way that was consistent with untransfected cultures,
although the vector itself seemed to decrease basal PAI-1 protein
levels (top two panels, Fig. 5). In BAECIIIR OE cells,
both TGF
1 and TGF
2 enhanced PAI-1 protein to essentially
equal extents (third panel, Fig. 5). In contrast,
BAECIIIR AS cells transfected with the type III receptor cDNA in the
reverse orientation were unresponsive to both TGF
isoforms
(bottom panel, Fig. 5). Notably, PAI-1 protein in the
untreated BAECIIIR AS cells was the highest among all the cell lines
tested.
Figure 5:
Expression of TGFIIIR increases the
effect of TGF
2 on PAI-1 protein in BAECs. Subconfluent cultures of
untransfected BAECs (Control), cells transfected with vector
alone (Vector), cells overexpressing type III receptor
(IIIR OE), and cells expressing an antisense construct of the
type III receptor (IIIR AS) were incubated with 0.5 ng/ml of
TGF
1 or 0.5 ng/ml of TGF
2. Equal amounts of protein from
detergent-extracted cell lysates were fractionated on a 10%
SDS-polyacrylamide gel, transferred to nitrocellulose membrane, probed
with antibodies to PAI-1 protein, and visualized by enhanced
chemiluminescence. Analogous results occurred in two separate
experiments.
Expression of Type III Receptor Protein Increases
Inhibition of Migration by TGF
Our laboratory
previously reported that TGF2 in BAECs
1 inhibited the migration rate of
BAECs, whereas TGF
2 had a very modest effect
(32) .
Therefore, we repeated migration assays with control and transfected
cells to see if there were any differences in the responsiveness to the
two TGF
isoforms. Similar to the earlier results, TGF
1, but
not TGF
2, inhibited migration in BAECs and in cultures transfected
with vector plasmid alone (Fig. 6). However, with BAECIIIR OE
cells, both TGF
1 and TGF
2 equally inhibited the migration
rates of these cells while BAECIIIR AS cells were refractory to both
TGF
isoforms. Previous studies
(32) also demonstrated
differences in BAEC proliferation responses to TGF
1 and TGF
2.
TGF
1 potently reduced proliferation of BAECs, whereas TGF
2
had no effect. In agreement with our PAI-1 and migration studies, when
we examined the proliferative response in BAECIIIR OE cells, the cells
were inhibited by both TGF
isoforms (data not shown).
Figure 6:
Expression of TGFIIIR increases the
inhibitory effect of TGF
2 on migration by BAEC. Migration assays
were performed with untransfected cells (BAEC), cells
transfected with vector alone (BAEC(V)), or cells transfected
with TGF
IIIR in the forward (BAECIIIR) or reverse
orientation (BAECIIIRAS). The cells were incubated with 0.5 ng
of TGF
1 or 0.5 ng of TGF
2 and assayed 6 days after removal of
the fences to allow migration to occur. Relative migration was analyzed
with the NIH Image Program.
Expression of the Type III Receptor Protein Increases the
Affinity of TGF
Our results so far indicated that the expression of
TGF2 for the BAEC Surface via the TGF
Type III
Receptor
type III receptor protein in BAECs increased their
responsiveness to TGF
2 by three separate criteria: proliferation,
PAI-1 protein, and migration assays. TGF
2 gave comparable results
to TGF
1. When an equal amount of unlabeled TGF
1 was combined
with
I-TGF
1, binding to the TGF
type III
receptor was not affected (Fig. 7, lane 2) whereas a
10-fold excess of unlabeled TGF
1 showed a 70-80% reduction
(Fig. 7, lane 3). Similar results occurred with
analogous amounts of unlabeled TGF
2, although it appeared to have
a slightly higher affinity for transfected type III receptor binding
sites (Fig. 7, lanes 4 and 5).
Figure 7:
TGF type III receptor actively
associates with TGF
1 and TGF
2. Subconfluent BAECIIIR OE cells
(lanes 1-5) were incubated with 100 pM
I-TGF
1, cross-linked with disuccinimidyl suberate
and analyzed as in Fig. 1A. During the incubation period with
iodinated TGF
1, labeling medium included no addition (lane
1), unlabeled TGF
1 at 100 pM (lane 2), or
1000 pM (lane 3); or unlabeled TGF
2 at 100
pM (lane 4) or 1000 pM (lane
5).
, respond well to TGF
1, but are relatively refractory
to TGF
2. Expression of transfected type III receptor protein
allowed BAECs to now respond to TGF
2 in terms of PAI-1 protein
levels, migration, and proliferation assays. These results indicate
that type III receptor protein expression increases the responsiveness
to TGF
2 in BAECs that do not normally respond to treatment with
TGF
2. Our binding studies agree well with previous observations
made by Wang and co-workers
(26) and Lopez-Casillas and
co-workers
(25) when they transfected type III receptor cDNA
into L
E
myoblast cell lines lacking the
TGF
type III receptor protein. In contrast, however, BAEC
transfectants containing the type III receptor cDNA in the reverse
orientation displayed a dramatic reduction in the surface expression of
both type I and type II receptors as assessed by radioligand binding
and cross-linking, and were refractory to both TGF
1 and TGF
2.
Therefore, our results indicate that complete down-regulation of the
TGF
type III receptor could alter the surface expression of the
TGF
type II and type I receptors in a fashion that is not yet well
understood.
2. TGF
2 has a
lower affinity for the cell surface type II receptor
(K
500 pM) than does
TGF
1 or TGF
3 (K
25-50 pM)
(5, 43) . One possible
explanation for this effect is direct physical interaction between the
type II and type III receptor proteins on the cell surface. Once
TGF
2 binds to the type III receptor it could then couple more
effectively to the type II receptors. Indirect evidence from our
studies with iodinated TGF
1 indicate that immunoprecipitation of
radiolabeled BAECIIIR OE cell lysates with antibodies to the type III
receptor did not co-precipitate much ligand-bound type II receptor.
However, immunoprecipitation with anti-type II antibodies
co-precipitated a significant amount of the type III receptor protein
as well as the type I receptor protein. Differences with these two
antibodies may reflect the overexpression of the type III receptor
protein relative to the type II and type I receptor proteins on the
surface of the BAECIIIR OE cells and suggest that a large fraction of
the type III receptors in this situation may not associate with the
other receptor types. Therefore, we suggest that there may be two kinds
of complexes: 1) complexes composed of TGF
, type II, and type I
receptors and 2) complexes composed of TGF
and all three cell
surface components. Expression of the type III receptor may
specifically promote TGF
2 access to type II receptor thus
overcoming the low affinity binding of TGF
2 to type II receptors.
It is also possible that TGF
2 binding to the type III receptor
elicits a conformation change in the TGF
2 molecule that more
favorably associates with type II receptor. Unbound TGF
1 might
approximate such a conformation better than unbound TGF
2. Recent
studies by Qian and co-workers
(44) , using domain swapping
experiments suggest that the different potencies of TGF
1 and
TGF
2 map to a 42-amino-acid region that includes the longest
-helix in the monomer. The availability of these chimeric TGF
molecules will enable us to identify which portion of the molecule is
responsible for interacting with the TGF
receptors. The recent
crystallography studies of TGF
2
(45, 46) will also
assist in our understanding of the important residues required for
interaction with the receptors.
type III receptor protein
(32) . BAECs lack the type III receptor
protein and are refractory to TGF
2 whereas rat fat pad capillary
endothelial cells contain the type III receptor protein and are
inhibited by TGF
1 and TGF
2 in proliferation
studies
(32) . Hence the differences in the surface expression of
the type III receptor protein in these two populations of endothelial
cells may well modulate their responsiveness to TGF
2. These data
are consistent with the concept that cells regulate their
responsiveness to TGF
isoforms by modulation of their TGF
receptors. Although the type III receptor does not appear to play a
direct role in signaling, recent findings from McAllister et
al.(47) suggest that defects in a related TGF
-binding
protein, endoglin, which does not have direct signaling activity as
well, is the cause of hereditary hemeorrhagic telangiectasia type I, an
autosomal dominant disorder that is characterized by multisystemic
vascular dysplasia and recurrent hemeorrhage. Hence although large
vessel endothelial cells lack the type III receptor, perhaps the type
III receptor plays an important role in early stages of vascular growth
in microvessel endothelial cells. Future experiments will be directed
toward down-regulating the expression of endogenous TGF
type III
receptor in cultured microvascular endothelial cells by using the
antisense construct to determine its effect in in vitro angiogenesis.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.