Endoglin Is an Accessory Protein That Interacts with the
Signaling Receptor Complex of Multiple Members of the Transforming
Growth Factor-
Superfamily*
Nadia Pece
Barbara
,
Jeffrey L.
Wrana§¶
, and
Michelle
Letarte
**
From the Cancer and Blood Research Program, and
§ Program in Developmental Biology, Hospital for Sick
Children, and Departments of
Immunology and
¶ Medical Genetics and Microbiology, University of Toronto,
Toronto M5G 1X8, Ontario, Canada
 |
ABSTRACT |
Endoglin (CD105) is a transmembrane glycoprotein
that binds transforming growth factor (TGF)-
1 and -
3, and
coprecipitates with the Ser/Thr kinase signaling receptor complex by
affinity labeling of endothelial and leukemic cells. The present study shows that in addition to TGF-
1 and -
3, endoglin interacts with activin-A, bone morphogenetic protein (BMP)-7, and BMP-2 but requires coexpression of the respective ligand binding kinase receptor for this
association. Endoglin cannot bind ligands on its own and does not alter
binding to the kinase receptors. It binds TGF-
1 and -
3 by
associating with the TGF-
type II receptor and interacts with
activin-A and BMP-7 via activin type II receptors, ActRII and ActRIIB,
regardless of which type I receptor partner is coexpressed. However,
endoglin binds BMP-2 by interacting with the ligand binding type I
receptors, ALK3 and ALK6. The formation of heteromeric signaling
complexes was not altered by the presence of endoglin, although it was
coprecipitated with these complexes. Endoglin did not interact with
BMP-7 through complexes containing the BMP type II receptor,
demonstrating specificity of its action. Our data suggest that endoglin
is an accessory protein of multiple kinase receptor complexes of the
TGF-
superfamily.
 |
INTRODUCTION |
The TGF-
1 superfamily
of structurally related peptides includes the TGF-
isoforms,
1,
2,
3, and
5, the activins and the bone morphogenetic proteins
(BMPs). TGF-
-like factors are a multifunctional set of growth and
differentiation factors conserved among flies, frogs, and mammals
(reviewed in Refs. 1-4). These factors control biological processes
such as embryogenesis, organogenesis, morphogenesis of tissues like
bone and cartilage, vasculogenesis, wound repair and angiogenesis,
hematopoiesis, and immune regulation (reviewed in Refs. 2 and 4-8).
Signaling by ligands of the TGF-
superfamily is mediated by a high
affinity, ligand-induced, heteromeric complex consisting of related
Ser/Thr kinase receptors divided into two subfamilies, type I and type
II (3). Formation of this high affinity complex is essential, as the
type II receptor transphosphorylates and activates the type I receptor
in a Gly/Ser-rich region (9-11). The type I receptor in turn
phosphorylates and transduces signals to a novel family of recently
identified downstream targets, termed Smads (12, 13).
Although the cooperativity between two kinase receptors is a general
signaling mechanism for the TGF-
superfamily, where the type I
receptors are considered the signal transducing receptors, ligand
binding ability is not restricted to receptor type. For TGF-
and
activin, the type II receptors T
RII and ActRII or ActRIIB, respectively, are known to bind ligand independently (14-16), while the corresponding type I receptors ALK5 (activin receptor-like kinase;
T
RI) or ALK4 (ActRIB) require the coexpression of the appropriate
type II receptors (9, 17-21). The BMP family differs in this respect
as the BMP type I receptors, ALK3 (BMPRI) and ALK6 (BMPRIB), can bind
BMP-2 and BMP-4 efficiently in the absence of the type II receptor, yet
require a type II receptor for transducing a transcriptional response
(22-26). In the case of BMP-7, the type II receptor BMPRII binds
ligand weakly, and cooperates with the type I receptors ALK3, ALK6, and
ALK2 (ActRI) to generate high affinity receptor complexes (24).
Furthermore, BMP-7 can also bind to the activin type II receptors and
form functional complexes with BMP type I receptors (27). This
cross-talk between the activin receptor system and the BMP receptors
suggests BMPs may have a broader function in vivo than first recognized.
Endoglin (CD105) is a homodimeric integral membrane glycoprotein
composed of disulfide-linked subunits of 90-95 kDa. In human, it is
expressed at high levels on vascular endothelial cells and on
syncytiotrophoblast of term placenta (28-30). It is transiently expressed on extravillous cytotrophoblasts and induced upon activation of peripheral blood monocytes (31, 32). Transient expression of
endoglin is also striking during human heart development, as it is
expressed at high levels on endocardial cushion tissue mesenchyme during heart septation and valve formation, and subsequently expression drops as the valves mature (33). Other sites of expression include a
population of pre-erythroblasts, leukemic cells of lymphoid and myeloid
lineages, and bone marrow stromal fibroblasts (28, 34, 35). Endoglin is
the target gene for the dominantly inherited vascular disorder
hereditary hemorrhagic telangiectasia type 1 (HHT1) (36). HHT is
characterized by frequent nose bleeds, mucocutaneous telangiectases,
and the development of arteriovenous malformations predominantly in
lung, brain, and the gastrointestinal tract that lead to recurrent
hemorrhage and shunting (37). We have recently shown that mutant forms
of endoglin are degraded intracellularly and that HHT1 is associated
with reduced levels of surface endoglin on endothelial cells and
activated monocytes (38).
Endoglin was shown to bind TGF-
1 and -
3 with high affinity, but
not -
2 (39), suggesting it mimics the isoform specificity of
T
RII. Endoglin was coprecipitated with T
RII and a type I receptor
in endothelial and leukemic cells (38, 40-42). Human umbilical vein
endothelial cells (HUVEC) were shown to have a single high affinity
binding site representative of T
RII complexes (39). Furthermore,
when overexpressed in U937 monocytic cells, endoglin did not alter
binding (42). These previous studies suggested that endoglin alone may
not bind TGF-
. We tested this using a COS1 transfection system and
now demonstrate that endoglin requires the coexpression of T
RII to
bind TGF-
1 and -
3. In addition, it binds activin-A, BMP-2, and
BMP-7 only in the presence of their respective ligand binding
receptors. We also demonstrate that endoglin associates with
heteromeric signaling receptor complexes of multiple members of the
TGF-
superfamily.
 |
EXPERIMENTAL PROCEDURES |
Cell Culture and Transfections--
Endothelial cells were
derived from HUVEC of newborns by previously published procedures and
maintained as described (28). NCTC2071 fibroblasts were cultured as
published (43). COS1 cells were maintained and transiently transfected
with expression constructs using the DEAE-dextran-chloroquine method as
reported (44, 45). Assays were performed 2 days after transfection.
Expression Vectors--
The EcoRI fragment of human full-length
endoglin in pcEXV-1 (46) was subcloned into pCMV5 (47) and used for
transient transfection, as all other cDNA used were already
subcloned into this mammalian expression vector. The pCMV5 expression
constructs containing cDNAs for T
RII, ALK5/HA (tagged at the
COOH terminus with the influenza hemagglutinin epitope, HA), ALK1/HA,
ActRII/HA, ActRIIB2/HA, ActRIIB2, ActRII/His
(tagged at the COOH terminus with six histidine residues), ALK6/HA,
ALK2/HA, and ALK3/HA have all been described previously (9, 16, 44, 45,
48, 49). BMPRII/FL (tagged at the COOH terminus with FLAG) was provided by F. Ventura, J. Doody, and J. Massagué (24).
Antibodies--
P3D1 and P4A4 hybridoma to human endoglin were
provided by E. A. Wayner (Seattle, WA) and were described and
characterized extensively (50). Murine IgG1 (Coulter
Electronics) was used as an isotype control for these two monoclonal
antibodies (mAb). For immunoprecipitation of T
RII, the polyclonal
antisera (pAb) C16, which was raised in rabbits by immunization with a
synthetic peptide corresponding to amino acids 550-565 of the highly
conserved carboxyl terminus of human type II TGF-
receptor, was used
(Santa Cruz Biotechnology Inc.). For immunoprecipitation of HA- and
FLAG-tagged TGF-
superfamily receptors, monoclonal antibodies 12CA5
(Boehringer Mannheim) and M2 (IBI, Eastman Kodak), respectively, were used.
Binding and Affinity Labeling--
TGF-
1 and TGF-
3 were
from R&D Systems. Recombinant human activin-A, BMP-2, and BMP-7 were
generous gifts from Y. Eto (Ajinomoto Co. Inc.), V. Rosen (Genentech
Institute), and K. Sampath (Creative Biomolecules) respectively.
TGF-
s, activin, and BMPs were iodinated with 125I using
chloramine-T as described previously (16, 51-53). For binding assays
with and without affinity labeling, HUVEC or transiently transfected
COS1 cell monolayers were incubated with 200 pM
125I-TGF-
1 for 4 h, washed, treated with or without
disuccinimidyl suberate (DSS; Pierce), and solubilized with lysis
solution (0.01 M Tris, pH 7.5, 0.128 M NaCl, 1 mM EDTA, 1% Triton X-100, and a mixture of protease
inhibitors) as published previously (54). Aliquots (300-500 µl) of
total cell lysates containing equivalent protein were subjected to
immunoprecipitation with control IgG (2 µg), anti-endoglin mAb P3D1
(4 µg), or polyclonal antibody C16 to T
RII (1 µg IgG). Immune
complexes were collected with Protein A- or Protein G-Sepharose
(Amersham Pharmacia Biotech), washed three times with lysis solution
containing 1% Triton X-100 (without protease inhibitors), and eluted
in 1% SDS (sodium dodecyl sulfate; >95 °C). When different
detergents were compared, lysis solution contained 1% digitonin (Wako)
or 1% CHAPS (Sigma) instead of Triton X-100; otherwise, the latter was
used throughout. Total lysates and eluted immunoprecipitates were
counted in a
counter (Beckman). Cross-linked receptors, bound to
radiolabeled ligand, were visualized by separation on SDS-PAGE (sodium
dodecyl sulfate-polacrylamide gel electrophoresis; 4-12% gradient
gels; Novex) under reducing (50 mM dithiothreitol) and
non-reducing conditions. Gels were subjected to autoradiography with
Kodak X-Omat film and DuPont Cronex-II screens or BioMax MS film and
the BioMax TranScreen HE intensifying screen system (Kodak). Multiple
exposures of each experiment were obtained.
For all other ligands tested, HUVEC or transfected COS1 were incubated
with 250 pM 125I-TGF-
3, 800 pM
125I-activin-A, 2 nM 125I-BMP-2, or
1-2 nM 125I-BMP-7 for 3-4 h, cross-linked
with DSS, and analyzed as described above using Triton X-100 in the
lysis solution. Conditions for immunoprecipitation and analysis were
also the same as above; however, some assays included the mAb P4A4 (1.6 µg) to endoglin, anti-HA mAb 12CA5 (1.5 µg), or anti-FLAG mAb M2 (3 µg) to tagged receptors.
Metabolic Labeling and Western Blot Analysis--
Endoglin and
T
RII expression in transfected COS1 cells were quantitated by
metabolic labeling in Fig. 1. Briefly, transfected COS1 were treated in
parallel to affinity labeling by incubation with 100 µCi/ml
[35S]Methionine (Met) (Tran35S-label; ICN
Pharmaceuticals Canada Ltd.) in Met-free Dulbecco's modified Eagle's
medium (low glucose; Life Technologies, Inc.) for 4 h,
solubilization in lysis solution containing 1% Triton X-100, and
immunoprecipitated with saturating amounts of antibodies, and
quantitated using a PhosphorImager and Image Quant Software (Molecular
Dynamics) according to published procedures (38). In other experiments,
endoglin protein levels in transfected COS1 cells were monitored by
Western blot analysis. Aliquots of total lysates from affinity-labeled
transfected cells were separated by SDS-PAGE (4-12% gradient,
non-reducing conditions) and assayed by immunoblotting using mAb P4A4
(1.6 µg/ml) to endoglin as described previously (38). For
determination of BMPRII/FL protein levels, aliquots of total cell
lysates were separated by SDS-PAGE and assayed by immunoblotting with
mAb M2 to FLAG as described (45). Immunoblots were visualized using the
enhanced chemiluminescence detection kit (ECL®; Amersham
Pharmacia Biotech) using the protocols provided. Multiple exposures
using Hyper-film (Amersham Pharmacia Biotech) were obtained.
Cell Surface Biotinylation--
Equivalent numbers of HUVEC or
transiently transfected COS-1 cells at subconfluence (~90%) were
surface-labeled with biotin in the absence of added ligand as reported
(38). Cells were lysed and immunoprecipitated as described under
"Binding and Affinity Labeling" except lysates were precleared for
at least 1 h with Protein A-Sepharose® CL-4B
(Amersham Pharmacia Biotech). Eluates were fractionated on 4-12%
SDS-PAGE gels, transferred to polyvinylidene difluoride nylon
membranes, and blocked as described for Western blots. Membranes were
probed with streptavidin-horseradish peroxidase (400-fold dilution in
Tris-buffered saline with Tween 20; Amersham Pharmacia Biotech) for 20 min, and biotinylated proteins were detected using ECL (Amersham
Pharmacia Biotech); multiple exposures were obtained.
 |
RESULTS |
Endoglin Requires the Coexpression of T
RII to Bind
TGF-
1--
Previous studies have shown that endoglin interacts with
the TGF-
binding complex. To investigate the nature of this complex, we transiently expressed endoglin in COS1 cells in the presence or
absence of T
RII. Cell monolayers were affinity-labeled with 125I-TGF-
1, chemically cross-linked using DSS, and cell
lysates fractionated by SDS-PAGE either directly or after
immunoprecipitation using antibodies to endoglin or T
RII (Fig.
1A). Compared with the vector
control, no affinity-labeled proteins were detectable when endoglin was
expressed alone (compare lanes 4-6 with
lanes 1-3), whereas T
RII expressed alone
bound TGF-
1 strongly (lanes 7-9). Control
experiments confirmed that endoglin was expressed at levels comparable
to that of T
RII (Fig. 1B). We previously showed that
endoglin is processed by COS1 cells and is expressed on the cell
surface in the absence of T
RII (38). In contrast, when endoglin was
coexpressed with T
RII, affinity-labeled endoglin migrating as a
monomer of 105-120 kDa was detected (Fig. 1A,
lane 11). Since endoglin and T
RII migrate
close to each other on reducing SDS-PAGE gels, affinity-labeled
proteins were also analyzed on non-reducing gels (lanes
14-17). Under these conditions, products characteristic of
dimers (180 kDa) and oligomers (>200 kDa) of endoglin were detected
(39) and could be immunoprecipitated using anti-endoglin (Fig.
1A, lane 15). Immunoprecipitation with anti-T
RII revealed that endoglin coprecipitated with T
RII (85-95 kDa) (Fig. 1A, lane 16). Thus we
conclude that endoglin is unable to bind TGF-
1 when expressed
alone.

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Fig. 1.
Endoglin requires coexpression of T RII for
binding to TGF- 1. A, COS1 transiently transfected
with empty vector (pCMV5), endoglin (END) and/or T RII
were affinity-labeled with 200 pM
125I-TGF- 1, cross-linked with DSS, and solubilized in
Triton X-100. Aliquots of total lysates each containing 20 µg of
total protein were fractionated on 4-12% SDS-PAGE followed by
autoradiography. Aliquots of lysates were immunoprecipitated with mAb
P3D1 ( END), pAb C16 ( T RII),
and control IgG1 as indicated. Arrows indicate the position
of monomeric endoglin (END) and T RII (RII)
separated under reducing conditions (R), and of endoglin
dimers, oligomers (OLIGO) and T RII when fractionated
non-reduced (NR). B, aliquots of COS1 cells
transfected in A were metabolically labeled with
[35S]Met and solubilized in Triton X-100. Lysates
containing equivalent cpm were immunoprecipitated with mAb P3D1
(lanes 1-4) and pAb C16 (lanes
5 and 6) and fractionated by SDS-PAGE using
reducing conditions. The left bracket indicates
monomeric glycosylated endoglin (END) and precursor in
lysates of cells expressing endoglin (lanes 2 and
4). Fully glycosylated RII and precursor bands are indicated
by the right bracket (lanes
5 and 6).
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We also analyzed the effect of chemical cross-linking on
125I-TGF-
1 binding to endoglin (Table
I). TGF-
1 binding to endoglin alone
was very low in the presence of the cross-linker DSS, and may represent
association of endoglin with the low levels of endogenous TGF-
receptors present on COS1 cells (see Fig. 1A,
lanes 1-3). However, in the absence of DSS, no
binding of TGF-
1 to endoglin was observed (Table I). When T
RII
was coexpressed with endoglin, strong binding of
125I-TGF-
1 to endoglin was observed in DSS-treated
cells, while binding was significantly less in non-cross-linked
samples. In contrast to endoglin, TGF-
1 binding to T
RII showed
little dependence on the chemical cross-linker. These data suggest that
the binding of TGF-
1 to endoglin is unstable in the absence of a
cross-linker.
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Table I
Effect of chemical cross-linking on 125I-TGF- 1 binding to
endoglin and T RII transiently expressed in COS1 cells
COS1 cells were transiently transfected with the indicated gene
constructs endoglin (END) and T RII in the pCMV5 vector. Two days
after transfection, cells were incubated with 200 pM
125I-TGF- 1, treated with or without DSS, solubilized with
Triton X-100. T RII and endoglin were immunoprecipitated with pAb C16
( T RII) and mAb P3D1 ( END), respectively, from aliquots of
total lysates containing equivalent total protein content.
Immunoprecipitates were eluted from Protein A- or G-Sepharose in 1%
SDS (>95%) and counted in a counter. Counts/min (cpm) above
background (IgG) for immunoprecipitates are reported.
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Interaction of Endoglin with TGF-
1·T
RII on HUVEC Is
Preserved in Digitonin--
We next examined the binding of
125I-TGF-
1 to HUVEC (Table
II), which express both T
RII and
endoglin. In Triton X-100, cpm eluted from anti-T
RII
immunoprecipitates showed little dependence on the presence of
cross-linker whereas, those eluted from anti-endoglin immunoprecipitates were dependent on cross-linker. Since detergents such as Triton X-100 can disrupt membrane protein complexes, we also
solubilized cells with milder detergents like CHAPS and digitonin known
to better preserve some protein/protein interactions (55). Of the three
detergents tested, CHAPS was most effective in preserving TGF-
1
interaction with T
RII. In CHAPS, we also recovered 12% of the
radiolabeled TGF-
1 in the anti-endoglin immunoprecipitates in the
absence of the cross-linker versus 4% recovery in Triton X-100 (Table II). In digitonin, however, we recovered 36% of the radiolabeled TGF-
1 in anti-endoglin immunoprecipitates relative to
cross-linked samples, which was comparable to 46% in the anti-T
RII immunoprecipitates, despite an overall reduced efficiency of lysis. These data suggest that TGF-
1 interaction with endoglin can be preserved in mild detergents.
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Table II
Effect of chemical cross-linking on 125I-TGF- 1 binding to
HUVEC monolayers and receptor complexes using different detergents for
solubilization of membrane proteins
HUVEC were incubated with 200 pM 125I-TGF- 1,
treated with or without DSS, and solubilized with Triton X-100, CHAPS,
or digitonin plus protease inhibitors. T RII and endoglin were
immunoprecipitated with pAb C16 ( T RII) and mAb P3D1 ( END),
respectively, from aliquots of total lysates containing equivalent
total protein content. Immunoprecipitates were eluted from Protein A-
or G-Sepharose in 1% SDS (>95%) and counted in a counter.
Counts/min (cpm) above background (IgG) for immunoprecipitates are
reported.
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Immunoprecipitation of T
RII·endoglin complexes using pAb C16
directed to the COOH terminus of T
RII consistently yielded efficient
coprecipitation of endoglin (see Fig. 1). In contrast, anti-endoglin
coprecipitated little T
RII that required overexposure of autorads
for visualization. Since these experiments were routinely performed in
Triton X-100, we determined whether CHAPS or digitonin might preserve
endoglin·T
RII complexes in anti-endoglin immunoprecipitates. HUVEC
were affinity-labeled with 125I-TGF-
1 and lysed in
Triton X-100, CHAPS, or digitonin, and the lysates subjected to
immunoprecipitation with antibodies directed against T
RII or
endoglin (Fig. 2). The profile of
endogenous receptor complexes immunoprecipitated from Triton
X-100-solubilized HUVEC (Fig. 2, lanes 4 and
5) was similar to that previously reported (38, 39) and was
comparable to that observed in COS1 cells coexpressing T
RII and
endoglin (see Fig. 1A). Similar results were obtained in
CHAPS-solubilized cells (lanes 6 and
7). However, in the presence of digitonin, we observed
efficient coprecipitation of affinity-labeled T
RII with the
anti-endoglin (lanes 9 and 15).
Furthermore, the relative intensities and profile of digitonin solubilized receptor complexes were similar in the anti-endoglin and
the anti-T
RII immunoprecipitates (compare lane
8 with lane 9, and lane
14 with lane 15). Thus digitonin
preserved endoglin association with both T
RII and TGF-
1. We have
shown that detergents that disrupt T
RII association with endoglin in
anti-endoglin immunoprecipitates also disrupt coprecipitation of
TGF-
with endoglin in the absence of cross-linker. Furthermore
digitonin, which preserved association of T
RII with endoglin, also
led to coprecipitation of TGF-
1 with endoglin in the absence of
cross-linker. Thus, the ability of endoglin to maintain interactions
with TGF-
1 is dependent on its association with T
RII. Based on
these results, we propose that endoglin is not itself a TGF-
receptor, but rather is cross-linked with the ligand through
association with the TGF-
type II receptor.

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Fig. 2.
The interaction of endoglin with TGF- 1 and
T RII is more stable in digitonin. HUVEC were affinity-labeled
with 200 pM 125I-TGF- 1, treated with DSS,
and solubilized with 1% Triton X-100 (T), CHAPS
(C), or digitonin (D) plus protease inhibitors.
Aliquots of total lysates were analyzed as in Fig. 1 (lanes
1-3). Detergent extracts containing equivalent total
protein content were immunoprecipitated with pAb C16
( T RII; lanes 4,
6, and 8) and mAb P3D1 ( END;
lanes 5, 7, and 9) and
fractionated non-reduced. Arrows indicate the
affinity-labeled endoglin (END) dimers, oligomers
(OLIGO), and the type II receptor (RII).
Fractionation of these samples using reducing conditions
(lanes 10-15) shows that the affinity-labeled
monomeric endoglin product(s) migrate slightly above RII. Traces of
receptor I (RI) are also noted.
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Endoglin Interacts with TGF-
3·T
RII and Requires
Coexpression of T
RII for Association with TGF-
3--
To
determine if endoglin could interact with T
RII bound to TGF-
3,
HUVEC were affinity-labeled using 125I-TGF-
3 and
analyzed by immunoprecipitation, SDS-PAGE, and autoradiography (Fig.
3A). Analysis of total cell
lysates and immunoprecipitates revealed specific cross-linking of
125I-TGF-
3 to T
RII and to endoglin dimers (180 kDa)
and oligomers (>200 kDa). This confirms observations that showed
competitive inhibition of TGF-
1 interaction with endoglin by the
TGF-
3 isoform (39). Endoglin dimers and oligomers were
coprecipitated by anti-T
RII, demonstrating that endoglin and T
RII
form a complex with TGF-
3 (Fig. 3A, lane
3). In COS1 cells, we next established that endoglin required the coexpression of T
RII for binding to TGF-
3 (Fig. 3B). No binding of TGF-
3 to COS1 cells transfected with
endoglin alone was observed despite high levels of endoglin expression as measured by Western blotting (Fig. 3B, lanes
1-4). However, when coexpressed with T
RII (Fig.
3B, lanes 5-8), endoglin bound to
TGF-
3 and could be immunoprecipitated with anti-T
RII, best seen
under non-reducing conditions (lanes 13-15).
Thus, endoglin interacts with either TGF-
1 or -
3 and requires
coexpression of T
RII to associate with these ligands.

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Fig. 3.
Endoglin interacts with TGF- 3·T RII
and requires coexpression of T RII for association with
TGF- 3. A, HUVEC were incubated with 250 pM 125I-TGF- 3, cross-linked with DSS,
solubilized with 1% Triton X-100, and analyzed as described in Fig. 1.
Total lysates containing 15 µg of total protein were fractionated
non-reduced (lane 1). Total binding was specific,
as binding in the presence of 40-fold excess competing unlabeled ligand
was 4000 cpm compared with 25,000 cpm/100-mm dish and revealed no
detectable receptors (lane 2). Cell extracts
containing equivalent total protein content were immunoprecipitated
with pAb C16 ( T RII; lane
3), mAbs P3D1 and P4A4 ( END; lanes
4 and 5, respectively), and fractionated
non-reduced. Arrows indicate the affinity-labeled endoglin
(END) dimers, oligomers (OLIGO), and the type II
receptor (RII). Fractionation of these samples using
reducing conditions (lanes 6-8) shows monomeric
endoglin affinity-labeled with TGF- 3 migrating slightly above
affinity-labeled T RII. B, COS1 cells were transiently
transfected as in Fig. 1 with pCMV5 empty vector, pCMV5-END, and/or
pCMV5-T RII as indicated. Two days after transfection, cells were
incubated with 250 pM 125I-TGF- 3,
cross-linked with DSS, solubilized with 1% Triton X-100, and analyzed
as described in Fig. 1. Total lysates that were fractionated reduced
are shown (lanes 1-4). Endoglin expression was
analyzed by Western blotting of an aliquot of these total lysates,
fractionated non-reduced, and probed using anti-endoglin mAb P4A4
(lower panel, lanes 1-4).
Aliquots of total lysates were immunoprecipitated with mAb P3D1
( END; lanes 5-8) and pAb C16
( T RII; lanes 9-12),
and fractionated reduced. Arrows indicate the
affinity-labeled monomeric endoglin (END) and T RII
(RII). Fractionation of these samples using non-reducing
conditions (lanes 13-15) reveals endoglin
dimers, oligomers (OLIGO), and T RII.
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Endoglin Interacts with Heteromeric Receptor Complexes Containing
ALK5 and Does Not Disrupt Their Formation--
ALK5 (T
RI)
preferentially interacts with ligand-bound T
RII to generate a
TGF-
receptor signaling complex. ALK5 is unable to bind TGF-
on
its own, but does so when coexpressed with T
RII (9). Having
established that endoglin recognizes ligand-bound type II receptors, we
investigated whether it could modulate binding and subsequent formation
of heteromeric complexes between ALK5 and T
RII (Fig.
4). No binding is observed when endoglin
and ALK5 are coexpressed (Fig. 4, lanes 1 and
2), but cotransfection of T
RII leads to binding of
TGF-
1 to both ALK5 and endoglin (lanes 3-5).
Anti-endoglin immunoprecipitated ALK5 and T
RII with endoglin (lanes 6 and 7). The anti-HA
immunoprecipitates showed that the heteromeric complex between ALK5 and
T
RII is not affected by endoglin expression (lanes
8 and 9). Together, these data show that endoglin
can interact with kinase receptor complexes containing ALK5, but does
not enhance overall binding, nor modulate the association of receptor I
with receptor II.

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Fig. 4.
Endoglin interacts with heteromeric receptor
complexes containing ALK5. COS1 cells were transiently
transfected, affinity-labeled with 200 pM
125I-TGF- 1, and analyzed as in Fig. 1. ALK5 was tagged
at the carboxyl terminus with HA. All samples were fractionated
reduced. Shown are total lysates (lanes 1-5) and
eluates from immunoprecipitates with mAb P3D1 ( END) and
with mAb 12CA5 ( HA) in lanes 6 and
7 and lanes 8 and 9,
respectively. Arrows indicate the positions of the
affinity-labeled products, endoglin (END), T RII
(RII), and the type I receptors (RI).
|
|
Endoglin Binds Activin-A or BMP-7 When Coexpressed with ActRII or
ActRIIB2--
As endoglin was cross-linked to TGF-
1 and
-
3 through its association with T
RII, we tested whether it could
interact with other type II receptors of the TGF-
superfamily. We
first examined activin-A, which binds to two related type II receptors,
ActRII and ActRIIB, and signals through a mechanism similar to that
defined for TGF-
receptors (44). COS1 cells were transiently
transfected with endoglin alone or together with ActRII or
ActRIIB2 (a ligand binding functional isoform of ActRIIB;
Ref. 16) and were then affinity-labeled using
125I-activin-A (Fig.
5A). When transfected alone,
endoglin did not bind activin-A, despite efficient expression of the
protein (Fig. 5A, lanes 1 and
2). However, in COS1 cells coexpressing type II receptors,
anti-endoglin immunoprecipitated 125I-activin-A
cross-linked to endoglin dimers and oligomers (lanes 12 and 13), and coprecipitated activin type II
receptors (lanes 10-13). Thus endoglin can form
complexes with ActRII or ActRIIB2 bound to activin-A. As
these receptors can also bind BMP-7 (27), we determined whether
endoglin might also associate with activin type II receptors bound to
BMP-7 (Fig. 5B). Endoglin alone did not bind BMP-7, but did
so in the presence of coexpressed ActRII and to a lesser degree with
ActRIIB2 (bottom panel). Reduced
efficiency of cross-linking of ligands to ActRIIB versus
ActRII has been reported (16).

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Fig. 5.
Endoglin binds activin-A or BMP-7 when
coexpressed with activin type II receptors. A, COS1
cells were transiently transfected with various combinations of pCMV5
empty vector, pCMV5-END, and HA-tagged pCMV5-ActRII or
pCMV5-ActRIIB2, affinity-labeled with 800 pM
125I-activin-A, and analyzed as in Fig. 1. Total lysates
that were fractionated reduced (R) are shown in
lanes 1-6; affinity-labeled type II receptors
(RII) are indicated by left bracket.
Endoglin expression was analyzed by Western blot of an aliquot of this
total lysate as in Fig. 3B (lower
panel, lanes 1-6). Eluates from
immunoprecipitates with mAb P3D1 ( END) were fractionated
reduced in lanes 7-11. Right
brackets indicate the positions of monomeric
affinity-labeled endoglin (END) and type II receptor
(RII). Corresponding eluates from lanes
10 and 11 were fractionated non-reduced
(NR) in lanes 12 and 13,
respectively, and lane 14 represents the negative
control for these conditions. Arrows indicate the position
of endoglin dimers and oligomers (OLIGO) and ActRII
affinity-labeled with activin-A. B, COS1 cells were
transiently transfected with various combinations of pCMV5 empty
vector, pCMV5-END, and/or pCMV5-ActRII/HA,
pCMV5-ActRIIB2/HA, or Flag-tagged BMPRII as indicated,
affinity-labeled with 1 nM 125I-BMP-7, and
analyzed as in Fig. 1. Total lysates fractionated under reducing
conditions (top panel) reveal affinity-labeled
type II receptors (RII). Endoglin expression was analyzed by
Western blotting of an aliquot of these total lysates as in Fig.
3B. BMPRII/FL was also analyzed by Western blotting of total
lysates using mAb M2 ( FLAG). Eluates from
immunoprecipitates with mAb P3D1 ( END) were fractionated
non-reduced in lower panel. Arrows
indicate the position of endoglin (END) dimers and oligomers
(OLIGO) affinity-labeled with BMP-7.
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|
Endoglin Does Not Interact with BMP-7·BMPRII Receptor
Complexes--
In these studies, BMP-7 was unable to interact with
BMPRII alone despite efficient expression of this receptor in COS1
cells (Fig. 5B). Coexpression of endoglin did not alter this
binding. Since binding of BMP-7 to BMPRII was shown previously to be
dependent on the coexpression of the type I receptors ALK6 or ALK2
(24), we investigated the ability of endoglin to interact with these complexes. Fig. 6 (A and
B) demonstrates binding of BMP-7 to ALK6·BMPRII or
ALK2·BMPRII complexes and is similar to that observed with ActRII or
ActRIIB2 (upper panels). However,
endoglin could not associate with the BMPRII complexes, while it could
with the ActRII and ActRIIB2 complexes (middle
panels). Indeed, under reducing conditions anti-endoglin
immunoprecipitates revealed coprecipitating affinity-labeled proteins
corresponding to the type II receptors, ActRII or ActRIIB2
together with the type I receptor, ALK2 (Fig. 6B,
lanes 9-12), but it did not alter the formation
of any of these complexes as seen in the anti-HA immunoprecipitates
(lower panels). Thus, endoglin is not found
associated with BMP-7·BMRPRII complexes containing ALK6 or ALK2.

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Fig. 6.
The type I receptors ALK2 or ALK6 neither
modulate nor induce endoglin interactions with BMP-7·ActRII, RIIB, or
BMPRII complexes. A, COS1 cells were transiently
transfected with pCMV5-ALK6/HA, with or without pCMV5-END, and/or
ActRII/His, ActRIIB2, BMPRII/FL as indicated,
affinity-labeled with 1 nM 125I-BMP-7 and
analyzed as in Fig. 1. Total lysates fractionated under reducing
conditions are shown in top panel, where
affinity-labeled type II receptors (RII) and ALK6
(RI) are indicated by arrows. Endoglin expression was analyzed by Western blot of an aliquot of these
total lysates as in Fig. 3B, shown just below top
panel. Eluates from immunoprecipitates with mAb P3D1
( END) were fractionated non-reduced in middle
panel. Arrows indicate endoglin (END)
dimers and oligomers (OLIGO) affinity-labeled with BMP-7.
Immunoprecipitation analysis of ALK6/HA ( HA) or BMPRII/FL
( FLAG) is shown in the lower panel,
and serves as the control for binding to and formation of heteromeric
complexes. Affinity-labeled type II receptors (RII)
co-precipitating with ALK6 (RI) are indicated with arrows.
B, COS1 cells were transiently transfected with
pCMV5-ALK2/HA, with or without pCMV5-END, and/or ActRII/His,
ActRIIB2, or BMPRII/FL as indicated and affinity-labeled
with 2 nM 125I-BMP-7. All samples were
processed as in A. In addition, anti-endoglin
immunoprecipitates were fractionated reduced (R) showing
coprecipitation of endoglin (END) with RII and ALK2
(RI) (right panel, lanes
9-12).
|
|
Endoglin Associates with BMP-2 When Coexpressed with the Type I
Receptors ALK3 and ALK6--
Unlike TGF-
, activin, and BMP-7, BMP-2
initiates signaling by first interacting with the type I receptors ALK3
or ALK6 and then recruits type II receptors into a signaling complex.
Since endoglin interaction with ligands appears to require expression of the ligand-binding component of the heteromeric Ser/Thr kinase receptor complex, we determined whether endoglin might interact with
BMP-2 in the presence of type I receptors. Endoglin alone was unable to
bind BMP-2, but did so upon coexpression with ALK3 or ALK6
(anti-endoglin (
END) panels, lanes
1 and 3 in Fig. 7, A and B). Interestingly, when endoglin was
coexpressed with either ALK3 or ALK6 and BMPRII, we observed a
substantial decrease in the association of endoglin with BMP-2 (Fig.
7A, compare lane 5 with
lane 3; Fig. 7B, compare
lane 6 with lane 3,
anti-endoglin (
END) panels). This occurred despite
efficient formation of BMP-2 binding receptor complexes in these cells
as seen in total lysates and anti-Flag and anti-HA immunoprecipitates,
and may reflect the inability of endoglin to associate with BMPRII, as
noted above.

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Fig. 7.
Endoglin binds BMP-2 when ALK3 or ALK6 are
coexpressed. A, COS1 cells were transiently transfected with
pCMV5-ALK3/HA, with or without pCMV5-END, and/or ActRII/His,
ActRIIB2 or BMPRII/FL as indicated and affinity-labeled
with 2 nM 125I-BMP-2. Analysis is the same as
in Fig. 6B. B, COS1 cells were transiently
transfected with pCMV5-ALK6/HA, with or without pCMV5-END, and/or
ActRII/His, ActRIIB2, or BMPRII/FL as indicated and
affinity-labeled with 2 nM 125I-BMP-2. Analysis
is the same as in Fig. 6B.
|
|
We also tested for the association of endoglin with BMP-2 in the
presence of ActRII and ActRIIB2. Consistent with previous observations on the Drosophila type II receptor, punt (56), we were unable to observe any binding of BMP-2 to ActRII (Fig. 7A, lane 8) or ActRIIB (data not
shown) when these receptors were expressed alone. However, in the case
of cells coexpressing ALK3 and either ActRII or ActRIIB2,
the association of endoglin with BMP-2 was comparable to that observed
with ALK3 alone (Fig. 7A, lanes 7 and
10 compared with lane 3). Similar
results were obtained in the case of cells coexpressing ALK6 and ActRII
or ActRIIB2 (Fig. 7B, lanes
8 and 10 compared with lane
3), although coexpression of ActRIIB2 yielded
lower overall levels of BMP-2 binding (seen in total lysates;
lanes 7-10); this may account for the reduced level of BMP-2 bound to endoglin observed in these transfectants. We
also confirmed that endoglin could associate stably with BMP-2 receptor
complexes containing the cotransfected ALK3 or ALK6 as they were
coprecipitated with anti-endoglin as seen under reducing conditions
(Fig. 7, A and B, lanes
11-13).
Endoglin Associates with the Ligand Binding Receptors--
Since
our data suggest endoglin interacts with ligand binding receptors, we
next tested whether endoglin could directly bind these receptors in the
absence of exogenously added ligand (Fig. 8). In COS1 cells that were transfected
with type II receptors, with or without endoglin, we analyzed receptor
interactions by surface biotinylation, solubilization in digitonin, and
specific immunoprecipitation. We found T
RII and ActRII
coprecipitated with anti-endoglin as seen under both non-reducing and
reducing conditions (Fig. 8A, lanes
3-6). This was not observed in BMPRII-expressing cells
(lanes 7 and 8) nor in the controls
(lanes 1 and 2). When the same
transfectants were analyzed with anti-T
RII (lanes
9-12), anti-HA (lanes 13-16), or
anti-FLAG (lanes 17-20), endoglin coprecipitated with T
RII, ActRII, but not BMPRII, respectively. Endoglin also interacted with ActRIIB2 (data not shown) in the absence of
added ligand. In a similar series of experiments, we found that the BMP
type I receptors, ALK2, ALK3, and ALK6, did not coprecipitate with
endoglin (Fig. 8B, lanes 3-16).
Furthermore, in COS1 cells expressing endoglin alone, we found no
evidence for interaction with endogenous receptors (lane
2). These data demonstrate that endoglin interacts
specifically with the ligand binding type II receptors T
RII, ActRII,
and ActRIIB2. However, association with type I receptors
may require ligand and/or chemical cross-linkers for detection by
coimmunoprecipitation. Furthermore, these latter interactions are not
likely dependent on the expression of endogenous type II receptors,
since the interaction of endoglin with the low level of any endogenous
type II receptors was undetectable both by affinity labeling and cell
surface labeling.

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Fig. 8.
In the absence of exogenous ligand,
endoglin associates with T RII and ActRII but not with BMPRII or the
BMP type I receptors. A, COS1 cells were transiently
transfected with pCMV5-T RII, ActRII/HA, BMPRII/FL, with or without
pCMV5-END as indicated and surface-labeled with NHS-LC-biotin,
solubilized with 1% digitonin, immunoprecipitated with mAbs P3D1
( END), 12CA5 ( HA), M2 ( FLAG),
and pAb C16 ( T RII), and eluates were
fractionated under non-reducing (top panels) and
reducing conditions (bottom panels) as in Fig. 2.
Gels were transferred to polyvinylidene difluoride nylon membranes,
probed with streptavidin-horseradish peroxidase, and detected by ECL.
Brackets indicate the positions of biotinylated
surface-expressed endoglin (END) dimers, oligomers
(OLIGO), and monomeric type II receptors (RII)
(non-reduced). Arrows indicate biotinylated monomeric
endoglin and the type II receptors (reduced). B, COS1 cells
were transiently transfected with pCMV5-ALK2/HA, ALK3/HA, ALK6/HA, with
or without pCMV5-END, and analyzed as in A. C,
HUVEC and NCTC2071 fibroblasts were surface-labeled with NHS-LC-biotin,
solubilized with 1% digitonin (left) or 1% Triton
(right), and analyzed as in Fig. 2. Arrows
indicate the positions of biotinylated endoglin dimers, oligomers, and
monomeric T RII (lanes 1-6; non-reducing
conditions) and monomeric endoglin and T RII (lanes
7-18; reducing conditions).
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|
We also investigated the interaction between endogenous endoglin and
T
RII, which are both expressed in endothelial cells and NCTC2071
fibroblasts (43). For this, the cells were surface-labeled by
biotinylation and solubilized in digitonin prior to immunoprecipitation (Fig. 8C). We observed that under non-reducing and reducing
conditions, endoglin coprecipitated with T
RII in the absence of
added ligand (compare lanes 2, 5,
8, and 11 with lanes 1, 4,
7, and 10). However, as with the affinity
labeling experiments (Fig. 2), this interaction was disrupted in Triton
lysates (Fig. 8C, lanes 13-18).
Together, these data suggest that endoglin associates with the type II
receptors in the absence of ligand.
 |
DISCUSSION |
The present studies show that endoglin can interact with TGF-
1,
3, activin-A, BMP-7, and BMP-2, but requires the coexpression of the
respective ligand-binding kinase receptor partner for binding and
specificity. For TGF-
1, its association with endoglin is better
demonstrated with the use of weak detergents that do not disrupt the
interaction of endoglin with T
RII. These results strongly suggest
that endoglin binds TGF-
secondarily to its association with T
RII
already bound to ligand. This would explain why the specificity and
affinity of endoglin for TGF-
isoforms mimics that of T
RII. It is
likely that this is true for all ligands that interact with endoglin.
Endoglin was first defined as a component of the TGF-
receptor
system when betaglycan was sequenced and found to be similar to
endoglin in particular, in the cytoplasmic tail where these two
proteins are 71% identical (39). Betaglycan is a proteoglycan (>200
kDa) also called a type III receptor that is required for presenting
TGF-
2 to the kinase receptor complex T
RII·ALK5, and promoting
signaling by this isoform (57). Betaglycan also binds other isoforms,
1 and
3, on its own and potentiates binding to cells ultimately
enhancing the response of cells to these ligands. Betaglycan acts as a
dual modulator of ligand access to the signaling receptors, as it can
be released from the cell membrane and binds ligand in soluble form;
thus, it is clearly defined as a receptor (58). No known signaling
domains have been identified in its structure; however, like endoglin,
its short cytoplasmic tail is highly conserved among species (43, 59).
Endoglin has often been compared with betaglycan and postulated to
function in a similar fashion by affecting the binding of TGF-
1 and
TGF-
3 to the signaling receptors (35, 39, 40, 60, 61), and it has
also been described as an auxiliary receptor (42). Our results clearly
demonstrate that endoglin does not function like the type III receptor
betaglycan, as it needs coexpression of a ligand binding receptor to
interact with ligand. Endoglin cannot bind ligand on its own; it does
not alter overall binding to the kinase receptors, but mimics the
specificity of the ligand binding receptor it interacts with. Indeed,
previous studies have shown that overexpression of full-length
functional endoglin in U937 does not alter the binding affinity of
receptor complexes (42), and we have obtained similar results when
endoglin is expressed in 3T3
fibroblasts.2 As we show
endoglin is not a true receptor, we define it is an accessory protein
that interacts with the ligand binding receptors of multiple members of
the TGF-
superfamily.
We demonstrate that endoglin not only interacts with ligand binding
receptors, but also associates with multiple heteromeric receptor
complexes (summarized in Fig.
9A). For TGF-
1,
3,
activin, and BMP-7, the type II receptors bind ligand and recruit the
type I receptor partners into a high affinity complex. Endoglin can interact with the ligand binding type II receptors, T
RII, ActRII and
ActRIIB2, regardless of which type I receptor partner is
coexpressed, and associates with these type II receptors in the absence
of exogenous ligand. Endoglin does not disrupt the formation of the signaling receptor complexes, and can be coprecipitated with these complexes. It could not, however, interact with BMP-7·BMPRII
complexes demonstrating specificity of endoglin action, nor did it
interact with BMPRII in the absence of ligand. However, the type I
receptors ALK3 and ALK6 bind BMP-2 and recruit the type II receptor
partners into a high affinity complex. Endoglin also interacts with
these ligand binding type I receptors and their respective type II
receptors (Fig. 9A). However, the association of endoglin
with ALK-3 and ALK-6 was only detectable in the presence of ligand and
a chemical cross-linker, which suggests these associations might be
more transient. Interestingly, we observed a reduction in BMP-2 binding to endoglin when BMPRII was coexpressed, yet the ligand binding to
BMPRII was not altered. Since endoglin probably binds ligands secondary
to its associations with the ligand binding receptors, these data
suggest that BMPRII may compete with endoglin for ALK-3 or ALK-6 and
that endoglin may be excluded from these complexes. Thus, endoglin is
an accessory protein that interacts with multiple heteromeric receptor
complexes containing TGF-
s, activins, and BMPs.

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Fig. 9.
Model of endoglin interaction with multiple
kinase receptor complexes of the TGF- superfamily.
A, summary of data demonstrating the ligands that endoglin
interacts with, the ligand binding receptors and the respective
partners present in the heteromeric complexes. B, the type
II (RII) and type I receptors (RI) are related
Ser/Thr kinase receptors, where RII is constitutively phosphorylated
(P) and transphosphorylates RI in the Gly/Ser-rich domain
(open circle) upon formation of a ligand
containing high affinity complex. For TGF- 1, 3, activin, and
BMP-7, the type II receptors bind ligand, whereas, for BMP-2, the type
I receptors ALK3 and ALK6 bind ligand. Endoglin interacts with these
receptors and becomes a component of the heteromeric receptor
complexes. This interaction is specific, as endoglin does not interact
with BMP-7·BMPRII. Endoglin does not alter the association of RI with
RII and thus interacts with a heteromeric complex that can initiate
downstream events. We postulate that endoglin might modulate signals,
by acting on known Smads and altering transcriptional responses or by
functioning in alternate pathway(s) leading to specific transcriptional
responses.
|
|
We have shown that endoglin interacts with specific ligand binding
receptors, and we postulate it is recruited into active receptor
complexes in this way (see model in Fig. 9B). What is the
significance of these findings? There are several levels of receptor
function that could be affected by the presence of endoglin. It could
modulate the activity of the receptor kinase complex. However, we have
found that endoglin does not alter the transphosphorylation of ALK5 by
T
RII.3 The receptor
complexes that endoglin interacts with contain the signal transducing
receptors ALK2, 3, 5, or 6. Since endoglin interacts with activin type
II receptors, it is likely to be found in heteromeric complexes
containing ALK4 (ActRIB) (Fig. 9). As multiple signal
transducing type I receptors interact with endoglin, it may function to
modulate receptor activation of downstream events, such as Smad
signaling (Fig. 9). For instance, endoglin might regulate BMP signaling
through Smads 1, 5, and 8 or TGF-
/activin signaling through Smads 2 and 3. Although endoglin expression has no effect on the induction of
the plasminogen activator inhibitor promoter by TGF-
or activin-A in
mink lung epithelial cells,3 a more comprehensive look at
downstream events may be warranted. Furthermore, endoglin could recruit
other proteins or a novel Smad into the signaling complex, thereby
inducing a specific nuclear response (Fig. 9).
Previous studies have shown that endoglin plays a role in the TGF-
pathway, as overexpression of endoglin modulates some but not all
TGF-
1 responses in U937 monocytes (42). Endoglin has also been
implicated in the regulation of trophoblast differentiation, a process
stimulated by activin and inhibited by TGF-
1 and -
3 (62, 63). As
we now show that endoglin can interact with activin type II receptors,
it might be functioning in activin as well as TGF-
receptor
complexes during placental development. Furthermore, activin and
TGF-
exert multiple effects on many cell types including erythrocytes, endothelial cells, stromal fibroblasts, and mesenchymal cells where endoglin is expressed (1, 64).
A major role for endoglin in the vasculature was inferred by the
finding that it is mutated in HHT1 (36). It is currently unclear what
molecular mechanisms underlie HHT pathology; however, our studies now
implicate pathways involving activins and BMPs, as well as TGF-
s.
Both TGF-
and activin are known to inhibit the proliferation of
endothelial cells in culture (65, 66). TGF-
is directly implicated
in vascular development and thought to control interaction between
endothelial cells and smooth muscle cells (67, 68). BMPs may also be
involved in these processes. BMP-2 and -7 can act on vascular smooth
muscle cells to inhibit their proliferation without stimulating
extracellular matrix synthesis, whereas activin-A has a
growth-stimulatory effect (69). The BMP-like factor GDF-5, which binds
ALK6, induces angiogenesis while BMP-2 does not (70). This is not
surprising, as vascular endothelial cells do not have specific binding
sites for BMP-2 (71). Together, these studies suggest a role for BMPs
in regulating the maintenance and/or formation of vasculature involving
both endothelial and smooth muscle cells where their specific response to these ligands depends on the receptors they express. The downstream effectors responsible for mediating these responses have yet to be
identified. In this context, it is interesting to note that ALK1 is the
target gene for HHT2 (72). Recently, ALK1 was shown to signal BMP-like
responses, yet its ligand in endothelial cells is unclear (73).
Furthermore, a class of inhibitory Smads were recently shown to be
expressed at high levels in endothelial cells during laminar shear
stress (74-77). As stress to blood vessels has been implicated in the
development of arteriovenous malformations (78), vascular Smads might
also play a role in the pathology of HHT. These findings, together with
the demonstration that endoglin is an accessory protein interacting
with multiple receptor kinase complexes, support the notion that HHT
could involve altered responses of the vasculature in pathways
additional to TGF-
. Elucidating the mechanisms of how endoglin
functions as an accessory molecule in the TGF-
superfamily is
critical to understanding the molecular mechanisms underlying the
development of HHT and biological processes where endoglin is expressed.
 |
ACKNOWLEDGEMENTS |
We gratefully acknowledge Y. Eto, V. Rosen,
and K. Sampath for generously providing us with activin-A, BMP-2, and
BMP-7, respectively. We thank F. Ventura, J. Doody, and J. Massagué for the BMPRII/FL construct; S. Vera for technical
assistance; M. Macías-Silva and S. Abdollah for helpful discussions;
and L. Attisano for insightful review of the manuscript.
 |
FOOTNOTES |
*
This work was supported in part by grants from the Medical
Research Council of Canada (MRC) and the National Cancer Institute of
Canada (NCIC).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. Section
1734 solely to indicate this fact.
MRC Scholar.
**
Terry Fox Research Scientist of the NCIC. To whom correspondence
should be addressed: Cancer and Blood Research Program, Hospital for
Sick Children, 555 University Ave., Toronto M5G 1X8, Ontario, Canada.
Tel.: 416-813-6258; Fax: 416-813-6255; E-mail:
mablab{at}sickkids.on.ca.
The abbreviations used are:
TGF-
, transforming growth factor
; BMP, bone morphogenetic protein; T
RII, TGF-
type II receptor; T
RI, TGF-
type I receptor; ALK, activin receptor-like kinase; BMPRII, BMP type II receptor; ActRII
and ActRIIB, activin type II receptors; ActRI and ActRIB, activin type
I receptors; BMPRI and BMPRIB, BMP type I receptors; RII, type II
receptors of the TGF-
superfamily; RI, type I receptors of the
TGF-
superfamily; HA, influenza hemagglutinin epitope; FL, FLAG
epitope; HHT, hereditary hemorrhagic telangiectasia; HUVEC, human
umbilical vein endothelial cells; DSS, disuccinimidyl suberate; pAb, polyclonal antibody; mAb, monoclonal antibody; PAGE, polyacrylamide gel
electrophoresis; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.
2
N. P. Barbara and M. Letarte, unpublished data.
3
N. P. Barbara and J. L. Wrana,
unpublished data.
 |
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