(Received for publication, September 22, 1994; and in revised form, November 28, 1994)
From the
Bone morphogenetic proteins (BMPs), which were originally
identified by their novel ability to induce de novo cartilage
and bone formation in vivo, are multifunctional proteins
structurally related to transforming growth factor-s, activins,
and inhibins. As a first step to elucidate the precise physiological
function as well as the action mechanism of BMPs, we have examined the
distribution of the specific cellular binding proteins for BMP-2 on a
wide variety of cell types. A single class of high affinity-specific
binding sites for BMP-2 were identified not only on osteoblastic cells
but also on major types of non-hematopoietic cells in a rather
ubiquitous fashion (1,200
60,000 receptors/cell, K
= 35
230 pM); these
cells included fibroblasts, keratinocytes, astrocytes, kidney
epithelial cells, and tumor cells of bone, muscle, lung, liver, kidney,
stomach, colon, prostate, and neuronal tissue. Other growth factors
including transforming growth factor-
, activin A, and
inhibin A did not compete for the binding of
I-labeled
BMP-2 to the cells. Affinity cross-linking of radiolabeled BMP showed
five components with apparent molecular masses of 170, 105, 90, 80, and
70 kDa common to all three fibroblast cell lines analyzed. On the other
hand, no specific binding sites for BMP-2 were identified on vascular
endothelial cells or on hematopoietic cells including RPMI 1788 and
RPMI 8226 (B-lymphocyte lineage), MOLT-3 and MOLT-4 (T-lymphocyte
lineage), HL-60 (myeloid lineage), and K-562 (erythroid lineage). These
results suggest that major types of cells other than hematopoietic
cells and vascular endothelial cells may be potential targets for BMP-2
action.
Bone morphogenetic proteins (BMPs) ()are a family of
proteins, which were originally identified and characterized by their
novel ability to induce cartilage and bone formation in ectopic
extraskeletal sites in vivo (Refs. 1 and 2 and reviewed in (3) ). Recently, several members of this protein family have
been isolated, cloned, and expressed as recombinant proteins; these
include BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, and BMP-7 (also referred to
as osteogenic
protein-1)(4, 5, 6, 7, 8) .
The BMPs are
30-kDa glycosylated proteins with disulfide-linked
dimeric structures(5, 6, 9) . Comparative
amino acid sequence analysis of the BMP family of proteins has
suggested that these molecules can be further divided into three
groups. The members of the first group, BMP-2 and BMP-4, are very
closely related to one another (
90% amino acid identity), but
their sequences differ significantly from those of BMP-3, -5, -6, and
-7. BMP-5, BMP-6, and BMP-7 exhibit
80% identity to each other,
thereby defining the second group. Of all the BMPs, BMP-3 is the most
distinct and by itself forms the third group(7) .
All the
BMPs contain the characteristic 7 highly conserved cysteines in their
carboxyl-terminal portions and thus belong to the transforming growth
factor- (TGF-
) superfamily, which includes TGF-
s,
activins, inhibins, and Müllerian inhibiting
substance (reviewed in (10) ). The members of TGF-
superfamily are multifunctional, e.g. TGF-
s are known to
be implicated in the regulation of a wide range of biological phenomena
such as cell proliferation, cell differentiation, tissue repair,
inflammation, angiogenesis, immunosuppression, and embryogenesis. In
this respect, expression of the BMP transcripts and presence of the BMP
proteins in various tissues including kidney, brain, and skin have been
reported(11, 12, 13) . Increasing evidence
suggests a regulatory role for BMPs in embryonic
development(14, 15, 16, 17) ,
indicating that the BMP family of proteins also has much broader
biological effects, unrelated to bone induction, on different cell
types. However, the precise physiological function as well as the
mechanism of action of BMPs are largely unknown.
As a first step to elucidate the biological action of BMPs, we have examined the distribution of specific cellular binding proteins (receptors) for BMP-2 on a wide variety of cell types and tissues; all the hormonally active polypeptides are believed to act on target cells by binding to specific cell surface receptors that are coupled to cytoplasmic signal transducers. In the present study, we have shown that the high affinity receptors for BMP-2 are present not only on osteoblastic cells but also on other types of cells in a rather ubiquitous fashion; these include fibroblasts, keratinocytes, astrocytes, kidney epithelial cells, and tumor cells of lung, liver, kidney, stomach, and neuronal tissue. On the other hand, cells of hematopoietic origin and vascular endothelial cells are shown not to express specific binding sites for BMP-2. In addition, we have characterized the specific cellular binding proteins for BMP-2 in several fibroblastic cell lines.
Binding of I-labeled BMP-2 to hematopoietic cells in
suspension (10
cells in 0.2 ml) was performed under
essentially the identical conditions as for the monolayer cells
described above. After the binding incubation, cells were pelleted,
resuspended in 0.2 ml of ice-cold binding buffer, and centrifuged
through a silicone oil/paraffin oil layer (0.2 ml); the radioactivity
associated with the resulting pellet was then
determined(24, 25) . Although all the hematopoietic
cells assayed in suspension lacked any specific binding sites for BMP-2
(see Fig. 5), this binding assay could be successfully used to
detect BMP binding proteins in PC12 and NB-1 cells and gave essentially
identical results as with the solid-phase monolayer assay described
above (data not shown).
Figure 5:
Binding of I-labeled BMP-2
to various lines of hematopoietic cells and human umbilical vein
endothelial cells. 83.3 (L) or 500 pM (H)
I-labeled BMP-2 was incubated with various types of cells
for 2 h at room temperature. Total binding (&cjs2110;) and nonspecific
binding (&cjs2112;) for each line of cells are shown; cell-associated
I-labeled BMP-2 radioactivities are normalized by
respective cell numbers and are expressed as cpm/10
cells.
The data represent mean ± S.D. of triplicate determinations.
Three independent experiments for each line of cells yielded similar
results.
Figure 1:
Time course of binding
of I-labeled BMP-2 to MC3T3-E1 cells. 333 pM
I-labeled BMP-2 was added to 30
10
MC3T3-E1 cells in 0.3 ml of binding buffer, and at the indicated
time intervals, cell-associated
I-labeled BMP-2 was
determined. At each time point, the binding of
I-labeled
BMP-2 was corrected for nonspecific binding, and the specifically bound
I-labeled BMP-2 radioactivity was plotted.
,
binding at 4 °C;
, binding at room temperature;
,
binding at room temperature in the presence of 0.1% NaN
.
Results are mean ± S.D. of triplicate determinations and are
representative of two independent
experiments.
Radiolabeled BMP-2 specifically
bound to MC3T3-E1 cells in a dose-dependent manner (Fig. 2). A
typical set of binding curves and corresponding Scatchard analysis are
shown in Fig. 2. These gave a linear plot characteristic of a
single high affinity binding site with 5,400 receptors/cell and a
dissociation constant (K) of 150 pM.
Figure 2:
Binding of I-labeled BMP-2
to MC3T3-E1 cells. Increasing concentrations of
I-labeled
BMP-2 were incubated with 31.5
10
MC3T3-E1 cells
for 2 h at room temperature, and the cell-associated radioactivity was
determined. The data are presented in a Scatchard plot, and the inset shows BMP-2 binding as a function of
I-labeled BMP-2 concentration where the BMP-2
specifically bound (
) is the difference between the total
(
) and nonspecific (
) binding. The data represent mean
± S.D. of triplicate determinations. Three independent
experiments yielded similar results.
The binding of I-labeled BMP-2 to other types of cells
(non-hematopoietic origin) was then analyzed. Representative Scatchard
plots for cells with low (
1,000), intermediate (
20,000), and
high (
60,000) numbers of high affinity receptors per cell are
shown in Fig. 3. The results of binding assays for other cell
lines using two different concentrations (83.3 and 500 pM) of
I-labeled BMP-2 are shown in Fig. 4. The number
and affinity of the receptors for BMP-2 on a variety of different cell
types are summarized in Table 1. Presence of the specific binding
sites for BMP-2 (with high affinity) was demonstrated on all the cell
lines assayed; these cells included fibroblasts, keratinocytes,
astrocytes, kidney epithelial cells, and tumor cells of bone, muscle,
lung, liver, kidney, stomach, colon, prostate, and neuronal tissue,
derived from different species (human, rodent, and dog) and from adult
and embryonic tissues. The relative high background of nonspecific
binding was observed for all cell types analyzed ( Fig. 2and Fig. 4), making it difficult to exclude the possible existence
of additional lower affinity sites in some cases since data in which
the nonspecific binding exceeded 50% of the total binding could not be
analyzed reliably. A similar high background of nonspecific binding was
reported for TGF-
(22) .
Figure 3:
Representative Scatchard plots of BMP-2
binding to cells expressing low, intermediate, and high numbers of
BMP-2 binding sites per cell. Increasing concentrations of I-labeled BMP-2 were incubated with 52
10
PAM212 cells (A), 10
10
TIG-3-20 cells (B), or 5
10
SF-TY cells (C) for 2 h at room temperature, and the
cell-associated radioactivity was determined. The data are presented in
Scatchard plots. Results shown are representative of two (A),
two (B), or three (C) independent
experiments.
Figure 4:
Binding of I-labeled BMP-2
to various types of non-hematopoietic cells. 83.3 (L) or 500
pM (H)
I-labeled BMP-2 was incubated
with various types of cells for 2 h at room temperature. Total binding
(&cjs2110;) and nonspecific binding (&cjs2112;) for each line of cells
are shown; cell-associated
I-labeled BMP-2
radioactivities are normalized by respective cell numbers and are
expressed as cpm/10
cells. The data represent mean ±
S.D. of triplicate determinations. Two or three independent experiments
for each line of cells yielded similar
results.
Figure 6:
Competition with growth factors for
binding of I-labeled BMP-2 to Swiss 3T3. 333 pM
I-labeled BMP-2 was incubated with 5
10
Swiss 3T3 cells in either the absence or presence of a
100-fold excess (33.3 nM) of the appropriate growth factor for
2 h at room temperature, and the cell-associated radioactivity (total
binding) was determined. Results are mean ± S.D. of triplicate
determinations and are representative of two independent experiments. EGF, epidermal growth factor; IGF-II, insulin-like
growth factor-II; aFGF, acidic fibroblast growth factor; bFGF, basic fibroblast growth
factor.
Figure 7:
Affinity labeling of SF-TY,
TIG-3-20, and Swiss 3T3 cells with I-labeled BMP-2.
SF-TY, TIG-3-20, and Swiss 3T3 cells were incubated with
I-labeled BMP-2 (50 or 250 pM) for 4 h at 4
°C in either the absence or presence of a 100-fold excess of
unlabeled BMP-2. Free ligand was removed, and bound
I-labeled BMP-2 was affinity cross-linked with
disuccinimidyl suberate. Samples were analyzed by SDS-PAGE followed by
analysis using a FUJIX Bioimaging analyzer BAS 2000. Arrowheads indicate positions of the cross-linked macromolecular components.
Results shown are representative of three independent
experiments.
In this paper, we have examined the distribution of the
cellular specific binding sites for BMP-2 on a large variety of cell
types. A complete Scatchard analysis was carried out for most cell
lines, but due to limitations in BMP-2 protein availability, some cell
lines were tested using only two different concentrations (83.3 and 500
pM) of I-labeled BMP-2. All of these results are
summarized in Table 1. The data show rather ubiquitous expression
of a single class of high affinity receptors for BMP-2 on the major
types of non-hematopoietic cells analyzed to date, which include bone,
muscle, skin, lung, liver, kidney, stomach, colon, prostate, and
neuronal cells, and suggest that all of these cell types may be
potential targets for BMP-2 action. In this respect, expression of the
BMP transcripts and presence of the BMP proteins in a wide variety of
tissues besides bone has been
reported(11, 12, 13) , and BMPs are suggested
to play important roles in the morphogenesis of the
embryo(14, 15, 16, 17) . Thus, it
seems very likely that BMP-2 and probably other BMP family members of
proteins have much broader biological effects on various cell types,
some of which are unrelated to bone formation. The precise
physiological function of BMPs on each of these cell types remains to
be elucidated. The presence of specific binding sites for BMP-4 on
MC3T3-E1 cells and NIH 3T3 fibroblasts(26) , PC12
cells(28) , and articular chondrocytes (29) has also
been recently reported.
On the contrary, hematopoietic cells
(B-lymphocyte lineage, T-lymphocyte lineage, myeloid lineage, and
erythroid lineage) and vascular endothelial cells were found not to
express any specific binding sites for BMP-2. All of the hematopoietic
cells we have analyzed in the present study are transformed cells;
although they are quite randomly selected, the observed absence of the
specific binding sites for BMP-2 on those cells might be the result of
malignant transformation. In this respect, however, expression of the
TGF- receptors on any given cell type has been reported to be
relatively unaffected by cellular transformation(22) . We have
obtained similar results with 3Y1 cells (Fischer rat embryo
fibroblasts) and various derived transformed cells such as SR-3Y1
(transformed by Rous sarcoma virus infection), HR-3Y1 (v-Ha-Ras
transfection), Py-3Y1 (mouse polyoma virus infection), SV-3Y1 (SV-40
infection), and NG-3Y1 (nitrosoguanidine treatment). Specific binding
of
I-labeled BMP-2 was demonstrated on all of these cells
in a one-point binding assay using 167 pM
I-labeled BMP-2, although degrees of specific
binding varied to some extent. We have also analyzed other cell lines,
such as Raji and RPMI 8866 for the B-lymphocyte lineage, Jurkat and
HUT-102 for the T-lymphocyte lineage, and U-937 for the myeloid
lineage, or phorbol 12-myristate 13-acetate-pretreated HL-60 and U-937
(which induces the differentiation of these cells into the
monocyte/macrophage lineage) (30) in the one-point binding
assay as described above. None of these cells shows any significant
expression of the specific binding sites for BMP-2. Furthermore, our
recent study on normal leukocytes prepared from the peripheral blood of
healthy human donors by Ficoll-Hypaque centrifugation does not reveal
any significant specific binding of BMP-2 to these cells. (
)In the meantime, Cunningham et al.(25) recently have reported that highly purified
populations of normal human monocytes possess specific binding sites
for BMP-4. Although all our results suggest that the major, if not all,
types of hematopoietic cells do not express any specific binding sites
for BMP-2, further detailed analysis may be necessary to draw a final
conclusion in this matter.
However, our finding that BMP-2 receptors
are absent from vascular endothelial cells and probably from
hematopoietic cells in adult animals seems interesting because all of
these cells are of splanchnic mesodermal origin. On the other hand, all
of the other cell types we have analyzed in the present study possess
specific binding sites for BMP-2, which include those developed from
ectodermal origin, endodermal origin, and mesodermal origin except for
the splanchnic lineage. The observed difference in the distribution of
specific binding sites for BMP-2 could be the result of diverged
expression of the BMP receptor gene(s) during embryonic development.
One speculation is that populations of cells that develop along the
splanchnic mesodermal lineage might be determined not to express
gene(s) coding for the BMP receptors. Such a distribution of BMP-2
receptors is apparently different from that of the TGF- receptors,
which are universally expressed on all cells of epithelial,
mesenchymal, and hematopoietic origin(22) . Thus, although
BMP-2 is suggested to have a wide spectrum of target tissues and cell
types for its action as discussed above, it is rather restricted as
compared with that of TGF-
.
Affinity cross-linking experiments
have revealed five macromolecular components that bind specifically to
BMP-2 in human and mouse fibroblasts. The apparent molecular masses of
these affinity-labeled complexes are 170, 105, 90, 80, and 70 kDa,
which are common to all the fibroblast cell lines analyzed and also to
PC12 cells. ()All of these cells have a single high affinity
receptor class, and thus it remains to be determined how the binding
sites identified by the equilibrium binding studies ( Fig. 2and Fig. 3and Table 1) relate to these five structural classes
of binding proteins identified by the affinity cross-linking studies (Fig. 7). Specific cellular binding proteins for BMP-4 with
apparent molecular masses of 164 and 54 kDa (in NIH 3T3 fibroblasts),
164 and 34 kDa (in MC3T3-E1 osteoblasts), and 165, 55, and 35 kDa (in
articular chondrocytes) have been recently
reported(26, 29) .
Although the receptors for BMPs
have not been well characterized, substantial progress has been
recently made on the characterization of receptors for other members of
TGF- superfamily such as TGF-
s and activins. For example, a
number of different size receptors and binding proteins for TGF-
s
have been identified in cultured cells and tissues by chemical
cross-linking experiments; these include type I (53 kDa), type II (75
kDa), type III (280 kDa), type IV (60 kDa), type V (400 kDa), and type
VI (180 kDa) receptors, as well as several other membrane binding
proteins of 40, 60, and 140 kDa (reviewed in (31, 32, 33) ). Among these receptors and
membrane binding proteins, the most widely distributed are the type I,
II, and III receptors. The roles of these different types of receptors
in the multiple functions of TGF-
s are unclear. However, several
lines of evidence suggest that the type I and type II receptors, both
of which have transmembrane serine/threonine kinase
structures(34, 35, 36) , are implicated in
the signal transduction of many effects of TGF-
s, while type III
receptors are involved in the presentation of the ligand to the
signaling receptors(31, 32, 33) .
The
presence of multiple cellular binding proteins for BMP-2 that we have
identified in the present study suggests that the functional receptors
for BMPs also are composed of several components. In this context, the
Daf-4 protein, a transmembrane serine/threonine kinase obtained from Caenorhabditis elegans, has been recently shown to bind
specifically to BMP-2 and BMP-4 and to form a 100-kDa cross-linked
complex(37) . The structure of Daf-4 is similar to that of the
activin type II receptor and TGF- type II receptor, and thus Daf-4
is suggested to be a type II receptor for BMPs. Very recently, when
this manuscript was in the final stage of preparation, ALK-3 and ALK-6
obtained from human and mouse, respectively(38) , and Brk25D
(the product of tkv gene) and Brk43E (the product of sax gene) from Drosophila(39, 40, 41) were suggested to be the
type I receptors for BMPs; these molecules bind specifically to BMP-4
or BMP-2 forming
80-kDa cross-linked complexes.
After
submission of this manuscript, isolation of a gene (brk-1)
encoding a putative type I receptor for BMPs from mouse fibroblasts was
reported(42) . Affinity labeling of brk-1 transfected
COS-7 cells with I-labeled BMP-2/BMP-4 was found to yield
two labeled products of
80 and
93 kDa; the
80-kDa band
is suggested to represent the product of brk-1 cross-linked to
the ligand monomer, while the
93-kDa band represents BRK-1
cross-linked to the ligand dimer. In this respect, some of our
cross-linking experiments with
I-labeled BMP-2 have
revealed an additional
155-kDa band other than the five components
described above in fibroblasts and PC12 cells.
Taken
together, these results might suggest that
I-labeled
BMP-2 has specifically bound to cellular proteins with molecular masses
of
140,
75 (this value corresponds to that of type II
receptor for TGF-
, and thus it could be a mammalian homologue of
Daf-4), and
52 kDa (this value corresponds to that of type I
receptor for TGF-
and thus it could be related to BRK-1/ALK-3 or
ALK-6); cross-linking of the ligand monomer (16 kDa) or dimer (30 kDa)
to each of these proteins would then generate the observed 170/155,
105/90, and 80/70-kDa species, respectively. Additional experiments are
needed to demonstrate whether the cellular specific binding proteins
for BMP-2 we have identified indeed constitute the functional BMP
receptor and how each of these molecules actually relates to Daf-4,
BRK-1/ALK-3, or ALK-6.