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J. Biol. Chem., Vol. 275, Issue 24, 17937-17945, June 16, 2000
From the Department of Biochemistry, University of Texas Health
Science Center, San Antonio, Texas 78284-7600 and the
Received for publication, August 24, 1999, and in revised form, February 3, 2000
Bone morphogenetic proteins (BMPs) occupy
important roles during development serving to direct cells through
specific differentiation programs. While several BMPs are essential for
embryonic viability, their significance in mediating intercellular
communication in the context of adult organ systems remains largely
unknown. In the adult rat we characterized the tissue- and
cell-specific transcription and translation of BMP-9. Utilizing a
ribonuclease protection assay, we determined that in the adult animal,
BMP-9 expression occurs predominantly in the liver. Furthermore, we
determined that the non-parenchymal cells of the liver,
i.e. endothelial, Kupffer, and stellate cells, are the
major sources of this message. Western analyses corroborate the
ribonuclease protection assay results, confirming that LEC and KC
contain an abundance of immunoreactive BMP-9. Using
[125I]BMP-9, a receptor with specific binding affinity
for BMP-9 was characterized in primary cultures of hepatic endothelial
cells and Kupffer cells. BMP-9 binding to these cell types was observed to be fully reversible and highly specific for this ligand.
Additionally, we demonstrate that BMP-9 is specifically internalized
upon binding to its receptor. This may represent a novel BMP receptor
and is the first to be characterized in primary cultures of mature
liver non-parenchymal cells. Our results depict BMP-9 as a potential autocrine/paracrine mediator in the hepatic reticuloendothelial system.
The secreted proteins of the transforming growth factor- BMPs and their receptors are expressed in numerous cell types and
during many different stages of embryonic development and adult life
(6, 7). These ligands are present in several soft tissues and skeletal
structures suggesting that their biology is more complicated than what
has so far been revealed by the phenotypes of the knockout animals now
available. BMP-2, -4, -5, -6, and -7 have been reported to be expressed
in major tissues of adult mammals including skin, heart, liver, kidney,
lung, and brain (6). BMP-7 is expressed abundantly in adult kidney and there is preliminary evidence that this BMP can obviate cell damage in
kidney resulting from ischemia (8, 9). While normal mice exhibit
epidermal expression of BMP-6 during both developmental and mature
stages of growth, adult mice overexpressing BMP-6 develop skin lesions
resembling psoriasis (10). Other than these reports, the role of BMP
signaling in mature soft tissue systems remains largely unexplored. It
is likely that the description of BMPs as differentiation factors is
incomplete since most adult tissues consist of terminally
differentiated cell populations. On the other hand, it is known that
most cell types undergo constitutive turnover, the significance of
which may not be fully appreciated beyond tissue damage-repair episodes.
With more than 20 mammalian BMPs now identified, only three type I
receptors and three type II receptors have been cloned in mammals which
have been shown to bind BMPs (11). The current receptor binding model
as described for BMPs and TGF- The receptor binding specificity of five BMPs have been described in
the literature and indicates that there is some promiscuity exhibited
by the known receptors for these ligands (13, 14). There is relatively
little published data describing cell-specific receptor binding
affinities and cell-specific receptor populations. There are a number
of publications documenting cross-linking of BMP ligands with their
receptors (13) and a select few reports characterizing the intrinsic
properties of BMP binding to both cell lines and primary cultures (15,
16).
We have investigated the tissue- and cell-specific expression of bone
morphogenetic protein-9 (BMP-9). BMP-9 was originally cloned from a
fetal mouse liver cDNA library and was shown to bind to specific
receptors on HepG2 cells (17). In addition, BMP-9 was shown to cause a
modest increase in proliferation of primary cultures of rat hepatocytes
(17). These findings prompted us to investigate the endogenous sources
of this cytokine and the cellular location of its receptor in livers of
adult rats.
This report describes the cellular expression and receptor binding
characteristics of bone morphogenetic protein-9. A survey of major
organs shows that in the adult rat, BMP-9 message predominantly occurs
in the liver. Within the liver, non-parenchymal cells, namely, Kupffer
cells (KC), hepatic stellate cells, and liver endothelial cells (LEC)
were used. In addition, the BMP-9 binding properties of both LEC and KC
were characterized. Our findings indicate that BMP-9 signals are
initiated via autocrine and/or paracrine mechanisms within the hepatic
sinusoid. To our knowledge, this is the first report of a BMP receptor
in the reticuloendothelial system of the liver.
Materials--
All reagents were biomedical research grade.
Collagenase (Type IV from Clostridium histolyticum),
protease (type XIV from Streptomyces griseus) (Sigma),
metrizamide (Accurate Chemical & Scientific Corp., Westbury, NY) and
Nycodenz (Sigma) for LEC and KC isolation by density gradient
centrifugation. Unlabeled BMP-2, -4, -6, -7, -9, and -12 were provided
by Genetics Institute Inc. (Cambridge, MA). BMP-2, -4, -6, and -12 were
shown to posses biological activity by Genetics Institute, Inc. BMP-12
was positive in the MLB13MYC clone 14 assay (18). BMP-2, -4, and -6 gave positive results in the W-20-17 assay (19). BMP-9 was iodinated as
described by Frolik et al. (20) and shown to possess
biological activity after iodination (17). TGF- Cell Isolation and Culture--
Rat liver sinusoidal cells were
isolated from 200 to 300 g adult Harlan Sprague-Dawley rats using
standard tissue digestion and centrifugal elutriation procedures.
Hepatic stellate cells were isolated as described by Rockey and Chung
(21). LEC were isolated by methods described by Gandhi et
al. (22). LEC were cultured on Corning 24-well cell culture plates
pre-coated with 33 µg of type I rat tail collagen/ml HBSS prior to
plating. LEC were maintained in RPMI 1640 supplemented with 20%
iron-supplemented fetal calf serum (Hyclone Logan, UT), 25 mM NaHCO3, 2 mM glutamine, 0.1 g of heparin/liter, and one 4.5-ml vial of penicillin (25,000 units)/streptomycin (25 mg/ml) per liter of media, pH 7.4. Initially, 2.5 × 106 LECs at a density of 2.5 × 106 cells/ml were placed in each well. Binding experiments
were performed on LEC within 1 day following establishment of the cells
in culture at 37 °C in a 5% CO2 atmosphere. KC were
isolated as described by Gandhi et al. (23). KCs were
cultured on Corning 24-well Cell Wells (Corning Inc., Corning, NY)
culture plates using RPMI 1640 (Life Technologies, Inc., Grand Island,
NY) media supplemented with 10% fetal bovine serum (Hyclone, Logan,
UT), 25 mM NaHCO3, and one 4.5-ml vial of
penicillin (25,000 units)/streptomycin (25 mg/ml) per liter of media,
pH 7.4. Initially, 2.5 × 106 cells at a density of
2.5 × 106 cell/ml were placed in each well. KCs were
cultured for 48 h at 37 °C in a 10% CO2 atmosphere
prior to conducting all receptor characterization experiments. Kupffer
cell and liver endothelial cell primary cultures are known to be highly
enriched (95%) for the specified cell type as determined by peroxidase
staining (KC marker) and acetylated low-density lipoprotein
incorporation (LEC marker) (24). Hepatocytes were isolated as described
by Seglen (25) and plated on Corning 24-well plates at 1 × 105 cell/well in William's E Medium (Life Technologies,
Inc.) supplemented with 10% fetal bovine serum (Hyclone) and 7 mg/liter insulin.
RNA and Protein Analysis--
Total RNA was purified form cells
and tissues using TRIzol Reagent and the manufacturer's protocol (Life
Technologies, Inc.) immediately following the cell isolation protocol.
The quantity of RNA extracted was estimated by determining the
A260 of the final precipitate dissolved in
diethyl pyrocarbonate water. Ribonuclease protection assays were
performed using RPA II Kit (Ambion Inc., Austin, TX). BMP-9 antisense
riboprobes were synthesized from a pGEM-3 construct containing 287 base
pairs of rat BMP-9 coding region generously provided by Genetics
Institute, Inc. Cyclophilin riboprobes were synthesized using
pTRI-cyclophilin-rat as a template (Ambion Inc. Austin, TX).
Cyclophilin and BMP-9 riboprobes were synthesized using Maxiscript
T7/T3 Kit (Ambion Inc.) with 800 Ci/mmol [ BMP-9 Binding, Cross-linking, and Binding Specificity--
BMP-9
binding assays were performed on 18-h cultures of primary hepatocytes
and liver endothelial cells and on 48-h primary cultures of Kupffer
cells. Cells were incubated for 1 h at 37 °C in binding buffer
(136.9 mM NaCl, 5.37 mM KCl, 1.26 mM CaCl2, 0.64 mM
MgSO4, 0.34 mM Na2HPO4,
0.44 mM KH2PO4, 0.49 mM
MgCl2, 25 mM HEPES, 0.5% bovine serum albumin,
pH 7.4) then transferred to binding buffer at 4 °C. Cells were
exposed to 35-90 pM [125I]BMP-9 with
appropriate concentrations of unlabeled BMP-9 for up to 24 h.
After equilibration, cells were washed three times with ice-cold
binding buffer then dissolved in cell solubilization buffer (25 mM HEPES, 10% glycerol, 1% Triton X-100, 1 mg/ml bovine serum albumin, pH 7.5) and counted. Cell numbers were determined from
video images taken of the cells just prior to solubilization using a
microscope-mounted video camera. Data were analyzed with Prism 2.0 software (Graphpad, Inc., San Diego, CA) and with the NIH program LIGAND.
The reversibility of [125I]BMP-9 binding was determined
by displacing the radioligand with excess unlabeled rhBMP-9. LECs were incubated at 4 °C overnight with 77 pM
[125I]BMP-9 alone or with 77 pM
[125I]BMP-9 plus an additional 8-fold excess unlabeled
BMP-9. Following the overnight incubation, the binding buffer was
exchanged for buffer containing only 615 pM unlabeled
BMP-9. At the appropriate time points, buffer as aspirated then the
cells were washed, dissolved, and counted.
Receptors were cross-linked to iodinated BMP-9 with 500 µM bis(sulfosuccinimidyl)-suberate (BS3),
(Pierce). KC were incubated overnight at 4 °C with 77 pM
[125I]BMP-9 alone or in the presence of 77 pM
or 1.9 nM unlabeled rhBMP-9. The cells were washed three
times to remove unbound ligand. Bovine serum albumin-free binding
buffer, with or without 500 µM BS3, was then
applied for 30 min at 4 °C. Cells were washed three times and
solubilized in cell lysis solution. Twenty-five µg of cellular
protein was resolved by 7.5% SDS-PAGE and the radioactive signal was
captured on a PhosphorImager plate.
The specificity of the BMP-9-receptor binding interaction was
determined by incubating KC and LEC with 77 pM
[125I]BMP-9 at 4 °C overnight alone or with an
additional 8- or 100-fold excess unlabeled competitor BMP. BMP-2, -4, -6, -9, and -12 were used in three separate experiments in 8-fold
excess of the radioligand and in one experiment at 100-fold excess of
the radioligand on both KC and LEC. BMP-7 was used in two experiments
at 8-fold excess of radioligand on LEC. BMP-7 was used only once on KC
at 8-fold excess and only once on KC at 100-fold excess (data not shown).
Receptor Internalization--
After a 1-h incubation interval in
serum-free media at 37 °C, cells were treated with 300 pM [125I]BMP-9 alone or with an additional
2.5 nM unlabeled BMP-9 for time periods up to 4.5 h at
37 °C. At the allotted times, cells were washed 3 times with
ice-cold serum-free media to remove unbound ligand, then washed 3 times
with ice-cold hypertonic acid wash solution (0.2 M acetic
acid, 0.5 M NaCl, pH 2.5) (27). Cells were solubilized with
cell lysis solution, acid wash-removable, and cell associated
radioactivity were determined separately. Data points showing the
internalized and cell surface radioactivity in the presence of 8-fold
excess of unlabeled BMP-9 represent results from a single experiment.
All data points with error bars represent pooled results from three
independent experiments.
Total RNA was isolated from the major organs of healthy, mature
Harlan Sprague-Dawley rats and probed for BMP-9 mRNA using a
ribonuclease protection assay (RPA) (Fig.
1). The housekeeping gene cyclophilin was
probed to indicate the equivalence of RNA loading as well as sample RNA
integrity. Among the organs and tissues examined, it is clear that in
the adult rat, BMP-9 transcription occurs predominantly in the
liver.
The major component cell populations of the rat liver were isolated and
the RPA was used to measure BMP-9 message levels (Fig. 2). Again, cyclophilin was probed to show
equal RNA loading and sample RNA integrity. BMP-9 message was found to
be expressed by KC, LEC, and hepatic stellate cells. Interestingly, we
were unable to detect BMP-9 message RNA in liver parenchymal cells.
Bone Morphogenetic Protein-9
AN AUTOCRINE/PARACRINE CYTOKINE IN THE LIVER*
, and
Genetics Institute, Inc.,
Cambridge, Massachusetts 02140
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ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(TGF-
)1 superfamily occupy
central roles in cellular differentiation and growth. Members of this
cytokine superfamily represent a highly conserved set of signaling
proteins whose analogs are distributed widely in organisms from
Drosophila melanogaster to Homo sapiens. A
specific subclassification within the TGF-
superfamily comprises the
bone morphogenetic proteins (BMPs). These secreted, dimeric proteins
were characterized originally by their ability to induce ectopic bone
formation following subdermal injection (1, 2). Generalizing from the
available literature, it appears that BMP signaling is important to an
organism during development and/or growth. For example, BMP-2 knockout
mice expire in utero with numerous developmental lesions on
extra-embryonic and embryonic structures (3). Mice deficient for BMP-7
die soon after birth exhibiting several bone and soft tissue
abnormalities which include underdeveloped kidneys that lack glomeruli
(4). BMP-5 knock-outs are healthy yet sustain many skeletal and soft
tissue defects including but not limited to, loss of one pair of ribs,
a smaller external ear, and a reduced ability to repair rib fractures
(5). These phenotypes arise when the affected tissues are undergoing morphogenesis suggesting that BMP signaling is particularly relevant during development when cells are being directed toward specific differentiation pathways.
has been recently reviewed (12) and
stipulates that BMP ligands bind type I and type II receptors with
similar affinity. However, signal transduction requires both types of
receptors. BMP ligands serve to bring type I and II receptors together.
This allows the Ser/Thr kinase activity of the type II receptor to
phosphorylate and activate the type I Ser/Thr kinase. The activated
type I receptor then initiates intracellular signaling by
phosphorylating cytoplasmic substrates known as SMAD proteins.
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EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 and
TGF-
3 were obtained from (R&D Systems, Madison, WI).
Two- to 4-month-old Harlan Sprague-Dawley rats (200-300 g) were the
source of the primary hepatic cell cultures and were handled in
accordance with regulations established by the NIH.
-32P]UTP
(NEN Life Science Products Inc., Boston, MA). The Pierce BCA protein
assay (Pierce) was used to estimate total cell protein concentration
from freshly isolated cell lysates prepared with cell lysis solution
(1.25 mM HEPES, 62 mM sucrose, 0.25% Triton X-100, 6 mM deoxycholate, 0.1 mM
phenylmethylsulfonyl fluoride, 0.078 mM pepstatin A, 0.004 mg/ml luepeptin, 0.012 mg/ml aprotinin). Total cellular proteins were
resolved by SDS-PAGE using a Tricine-based buffer system as described
by Schagger and van Jagow (26). Proteins were transferred onto
Immobilon-P polyvinylidene difluoride membrane (Millipore Corp.,
Bedford, MA) and subsequently immunoblotted using a BMP-9 monoclonal
antibody raised in mouse kindly provided by Genetics Institute, Inc.
Detection of the antigen-antibody complex was performed using the goat
anti-mouse IgG (H+L)-HRP conjugate secondary antibody (Bio-Rad) and ECL
(Amersham Pharmacia Biotech). Membranes were exposed to Hyperfilm
(Amersham Pharmacia Biotech) to obtain the desired intensity.
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RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

View larger version (68K):
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Fig. 1.
30 µg of total RNA
from major organs taken from 2-4-month-old Harlan Sprague-Dawley rats
was subjected to RPA using riboprobes for both BMP-9 and the
housekeeping gene cyclophilin. Probes were hybridized to yeast
tRNA and treated with RNase A/T1 (+) or not (
) to indicate probe
integrity and RNase activity. A, lane contents
from left to right: liver, bone, (femur) skin,
cartilage (xyphoid process), large (L) intestine, small
(S) intestine, lung, skeletal muscle, spleen. B,
lane contents from left to right:
liver, pancreas, brain, kidney, heart, stomach.

View larger version (67K):
[in a new window]
Fig. 2.
Liver fractionation was performed and total
RNA from cell fractions enriched for non-parenchymal and hepatocyte
cell types was analyzed by RPA. Antisense riboprobes for BMP-9 and
cyclophilin were used to probe 20 µg of total RNA from the following
cells: liver non-parenchymal cells (NC); hepatic stellate
cells (HSC); LEC; KC; hepatocytes (H). Yeast tRNA
was incubated with both probes and treated with RNase (A/T1) (+) or not
(
) to indicate probe integrity as well as RNase activity.
The presence of BMP-9 messenger RNA suggests that these cells are
capable of translating BMP-9 protein. To confirm this assumption, Western blot analyses were performed on whole liver homogenate and LEC,
KC, and hepatocyte cell lysates using a monoclonal antibody raised
against recombinant human BMP-9 (Fig. 3).
Freshly isolated cells were solubilized and samples of total cell
proteins were resolved by SDS-PAGE under reducing conditions. As a
control, 2 ng of rhBMP-9 was positioned in the first lane on
the left and is indicated by the band at approximately 13 kDa, the expected molecular mass of monomeric BMP-9. Hepatocytes did
not contain a detectable amount of the protein, however, BMP-9 is
associated with KC and LEC following their isolation from the liver
(Fig. 3, lanes LEC and KC). This monoclonal
antibody clearly detects BMP-9 protein in samples of liver
non-parenchymal cells (Fig. 3, lane NC). However, this
method was not sensitive enough to detect BMP-9 protein in samples
prepared from whole liver (Fig. 3, lane L). The
immunoreactive bands with masses greater than 13 kDa are due entirely
to secondary antibody immunoreactivity independent of the primary
antibody (data not shown). The Western blot analysis corroborates the
RPA results, establishing hepatic non-parenchymal cells as the primary
source of BMP-9.
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It is thought that BMPs act locally by autocrine and/or paracrine
mechanisms. Upon finding that liver cells express BMP-9 we sought to
identify a hepatic cell type capable of specifically binding this
cytokine. Primary cultures of LEC and KC bound iodinated rhBMP-9
([125I]BMP-9) as demonstrated by its displacement by
unlabeled BMP-9 (Fig. 4, A and
B). When cells were incubated at 4 °C in the presence of
35-90 pM [125I]BMP-9 and increasing
concentrations of unlabeled, homologous competitor, the resulting
competition binding isotherms depict a ligand-receptor interaction of
high affinity. Our assay was unable to detect [125I]BMP-9
binding in primary cultures of hepatocytes (data not shown).
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Binding parameters were similar between the two non-parenchymal cell types (Table I). LEC had an EC50 value of 57.2 pM with 95% confidence intervals ranging from 24.8 up to 131.9 pM. The EC50 value determined for KC was 5.1 pM with upper and lower 95% confidence intervals at 7.1 and 3.6 pM, respectively. We then used the NIH program LIGAND to construct Scatchard plots and to estimate apparent Kd and Bmax values, (Fig. 5, A and B).
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Initially, we were unable to fit the combined Kupffer cell data using
this program. Data for each experiment, when processed individually, yielded an extremely low apparent Kd
at 7.4 ± 11.5 attomolar (mean ± S.D., n = 6). Subsequently, we were able to fit the combined Kupffer cell data
using a cooperativity setting of
0.1 which yielded an apparent
Kd of 24.9 pM and
Bmax of 0.71 pM (Table I). In a
0.25-ml system, this represents 0.18 fmol/105 cells, or
approximately 1100 receptors/cell. Visual inspection of Fig.
5A shows that specific binding is less than 1 fmol/well.
Combined data for experiments using liver endothelial cells yielded an
apparent Kd of 84.6 pM and
Bmax of 2.41 pM (Table I). In a
0.25-ml system, this represents 0.63 fmol/105 cells, or
approximately 3600 receptors/cell. Introducing a cooperativity factor
of
0.1 made no significant difference to the fit of the liver
endothelial cell data. Visual inspection of Fig. 5B shows that specific binding is approximately 1 fmol/well.
Scatchard analysis assumes that ligand binding is reversible. In order to confirm that this was occurring in our system LEC were incubated in the presence of 77 pM [125I]BMP-9 at 4 °C for increasing amounts of time after which the amount of ligand bound was determined (Fig. 5C). It was shown that [125I]BMP-9 binding by LEC increased over the 6-h interval and that unlabeled BMP-9, when added in excess, was able to displace the radioligand (Fig. 5D).
We performed affinity label cross-linking experiments with the
radioligand. Binding equilibrium was established with primary cultures
of KC and the primary amine-targeting cross-linking reagent BS3 was applied. The cellular proteins were then
solubilized and resolved by SDS-PAGE under reducing conditions. The
radioactive signal was imaged with a PhosphorImager plate (Fig.
6). A major band with an apparent
molecular mass of 70-75 kDa is clearly evident. We interpret this band
to be a ligand-receptor complex consisting of a monomer of
[125I]BMP-9, 13 kDa in size, cross-linked to a
60-kDa
receptor. The signal at the bottom represents monomeric
[125I]BMP-9 that was not cross-linked. In addition, the
radiolabeled ligand-receptor complex was not seen when the indicated
concentrations of unlabeled rhBMP-9 were added. Finally, the formation
of the radiolabeled complex is dependent upon the presence of
BS3. The experiment was performed twice yielding identical
results.
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To demonstrate the specificity of BMP-9 binding to LEC and KC we
attempted to compete for the [125I]BMP-9-binding site
with other members of the TGF-
superfamily (Fig.
7). Using 2 ng/ml radioligand in an
overnight incubation at 4 °C, we established maximum binding for
both LEC (Fig. 7A) and KC (Fig. 7B). Competitor
BMPs-2, -4, -6, -9, and -12 were used at 16 ng/ml (8-fold excess,
filled bars) and 200 ng/ml (100-fold excess, open
bars). Only BMP-9 was able to compete for radioligand-binding sites on LECs and KCs. Comparing the amount of bound radioactivity in
the presence of 16 ng/ml to that bound in the presence of 200 ng/ml
unlabeled BMP-9, it was apparent that an 8-fold excess of BMP-9
established the nonspecific binding of [125I]BMP-9 to
both of these cell types. Both concentrations of unlabeled BMP-9
reduced the amount of bound radioactivity by nearly 80%. When
candidate competitor BMP-2, -4, -6, and -12 were provided at 100-fold
excess of the BMP-9 radioligand, binding was reduced by approximately
10%. Preliminary results obtained using excess BMP-7 as a competitor
have yielded similar results, suggesting that it is also unable to
compete for the BMP-9 binding site on these cell types (see
"Experimental Procedures" and data not shown). The BMPs tested here
are clearly not interacting with the same cell surface-binding site as
BMP-9. Therefore, in the interest of conserving reagents, this
experiment was repeated three times using competitor candidate BMPs and
TGF-
1,3 at 8-fold concentrations relative to the BMP-9
radioligand. The fact that none of these superfamily members were able
to compete for the [125I]BMP-9 binding sites suggested
that BMP-9 was interacting with a previously unidentified receptor on
these cells.
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We asked whether or not these cells could internalize the cell surface receptor-BMP-9 complex. Initially we determined that three acid wash cycles effectively remove more than 99% of the total BMP-9 bound (data not shown). In addition, we established that ligand internalization was temperature-dependent and that this process was minimal (approximately 10% bound radioactivity was internalized) when cells were incubated at 4 °C. LEC were incubated at 4 °C for 4 h in the presence of increasing concentrations of [125I]BMP-9. Acid wash-removable, internalized, and total bound [125I]BMP-9 were then determined (Table II)(see "Experimental Procedures"). A majority of the iodinated ligand remained on the cell surface when cells were maintained at 4 °C.
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To examine whether or not BMP-9 receptors internalize this ligand under
physiological conditions LECs were incubated at 37 °C in the
presence of 300 pM [125I]BMP-9 for intervals
up to 6 h. At the allotted times, acid wash-removable and cell
associated radioactivity were determined (see "Experimental Procedures"). Acid-washable radioactivity representing the
[125I]BMP-9 bound to cell surface receptors initially
increased as receptors were loaded with ligand and then decreased as
the cell surface receptor population was reduced (Fig.
8A). Cell associated radioactivity representing internalized [125I]BMP-9 also
increased initially, then superseded that which was on the cell surface
and finally reached a plateau (Fig. 8A). It was apparent
that LEC actively bound and internalized this ligand under these
culturing conditions.
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When this experiment was performed in the presence of an 8-fold excess of unlabeled ligand, the amount of cell surface and cell internalized radioactivity was reduced (Fig. 8A). This experiment demonstrated the specificity of the receptor-ligand internalization event.
Also, this experiment was performed on primary cultures of KCs (Fig. 8B). In short, KC have the capacity to bind and internalize [125I]BMP-9 as well. As the incubation time increased, more radioactivity became refractory to the acid-wash procedure (Fig. 8B). The experiment was performed with KCs in the presence of 8-fold excess unlabeled BMP-9 to show that the internalization event was specific for this ligand (Fig. 8B).
The conditioned media from the internalization experiments discussed
above were analyzed for metabolized fragments of
[125I]BMP-9. Aliquots of serum-free conditioned media
from LECs and KCs were resolved by SDS-PAGE. A single band of
radioactive signal was detected that was indistinguishable from the
signal produced by stock radioligand not exposed to cells. This
observation indicated that digested fragments of
[125I]BMP-9 were not a significant constituent in the
conditioned media.
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DISCUSSION |
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The initial study indicating that BMP-9 exhibits defined agonist
responses in liver cells (17) suggested a myriad of questions to us
concerning the origin and binding specificity of this TGF-
superfamily member. A survey of major organs revealed that the liver
possesses a robust ability to synthesize this putative intercellular signaling molecule. This finding is interesting when considering the
known expression patterns of other BMPs in adult mammals. For example,
BMP-6 is expressed by muscle, lung, brain, skin, and gut (28). Numerous
adult tissues have the capacity to express BMP-4 including spleen,
lung, kidney, liver, and heart (6).
Other members of the TGF-
superfamily have interesting
tissue-restricted expression patterns. In developing as well as adult mammals, GDF-8 is expressed exclusively by skeletal muscle (29). Removing this gene gave rise to an animal with substantially increased skeletal muscle mass, indicating that this cytokine may regulate muscle
growth (29). Of numerous adult organs surveyed for BMP-8A expression
(30), only cells of both male and female reproductive systems
transcribe this gene (31). In accordance with its organ-specific expression pattern, BMP-8A was found to be important in the maintenance of spermatogenesis in males (31).
Our results immediately suggest a liver-specific function for BMP-9. The fact that liver non-parenchymal cells reserve the capacity to synthesize and to bind this cytokine implies that BMP-9 signals mediate reticuloendothelial system function within the liver. The cells comprising this system represent three functionally unique cell populations representing a multifaceted support system to the liver parenchyma. Our results suggest that there is an ongoing, basal level of transcription and translation of BMP-9 by the sinusoidal cell populations.
In accordance with the hypothesis that BMPs operate via autocrine
and/or paracrine signaling mechanisms, we found a BMP-9 receptor in
primary cultures of KC and LEC. This receptors affinity for BMP-9 is in
the range previously reported for other members of the TGF-
superfamily (32). While there is abundant information available
depicting BMP binding and cross-linking to transformed cells
transfected with cloned receptors (13, 14, 33-35), there is relatively
little published information explicitly defining BMP binding affinities
or receptor populations in primary cell cultures. Primary human
monocytes respond to BMP-2B receptor binding with chemotactic activity
utilizing as few as 750 receptors per cell (15). BMP-4 has been shown
to specifically bind primary bovine chondrocytes
(Bmax 6000 rec/cell) and up-regulate
extracellular matrix protein synthesis in these cells (16). A rather
extensive survey of cell lines capable of binding BMP-2 shows that this BMP has picomolar affinity for receptors on several cell types (32).
The BMP-9 receptor binding activity and cell surface population appears to be of sufficient affinity and abundance to suggest physiological relevance. When analyzing BMP-9 binding to two different cell populations, similar binding parameters were found. KCs have an apparent Kd for BMP-9 that is only slightly less than that measured for LEC. KCs have fewer receptors/cell than LECs, e.g. the Bmax for KC appears to be approximately one-third of that found for LECs. In order to obtain the best fit of our data in Scatchard format, we found that it was necessary to invoke a negative cooperativity binding model in the Kupffer cells. The same model applied to liver endothelial cells did not significantly improve the quality of the fit. The biological implications of this observation will require more experimentation.
We were able to visually image this receptor in a covalently linked complex with the radioligand. When the molecular mass the BMP-9 monomer is subtracted, we calculate the molecular mass of the receptor to be in the range of 60-63 kDa. This is slightly larger than the known BMP type I receptors, ALK-2, -3, and -6 which have been shown to be 53-58 kDa in size (33, 34). There is not a clear signal at the expected size for a type II receptor-ligand complex. Our data suggest that this band is likely to be a type I Ser/Thr kinase.
The specificity of the ligand-receptor interaction is a critical
parameter that must be established in characterizing a relevant cytokine-receptor interaction. The mature TGF-
/BMP is a homodimer of
13-25-kDa subunits with 30-50% primary sequence homology (36). The
fact that BMP-2, -4, -6, -7, -12, TGF-
1, and
TGF-
3 were unable to displace [125I]BMP-9
indicates that this binding site is highly selective for BMP-9. Taking
our binding specificity and cross-linking data together with the
binding promiscuity and molecular weights of the known type I and II
receptors, it is our contention that a novel BMP receptor has been
identified in Kupffer cells.
Characterizing the cellular response to BMP-9 involved the investigation of potential cellular mechanisms involved in the processing the receptor bound cytokine. It is a common biological phenomenon for receptors of many different signaling pathways to be internalized upon binding their ligands. Among the possible consequences of receptor internalization is a change in the ability of the cell to respond to the ligand for that receptor. Some receptor/ligand systems, i.e. G protein-coupled and tyrosine kinase receptors, are subjected to this process as a means to turn off the signaling pathway or to remove the ligand from the receptor therefore rendering the receptor available for future signaling events (37, 38).
Transforming growth factor-
is internalized and metabolized by some
cell types (20, 39). A single study has been published documenting BMP
receptor internalization in skeletal muscle cells (40). Our results
show that BMP-9 receptors are internalized in the presence of ligand,
suggesting that the receptor can be down-regulated by its ligand.
Indeed, beyond the 2-h time point, the amount of radioactivity inside
both cell types exceeds that which remains on the cell surface. The
available data suggest that BMP-receptor internalization in the
presence of ligand may be a general mechanism used by BMP
receptor-containing cells to process BMP signals.
It is known that TGF-
isotypes 1-3 as well as other BMPs including
BMP-6 are expressed by mammalian liver (28, 41-43). TGF-
is
considered to be a major factor in the progression of hepatic fibrotic
and regenerative processes (44, 45). In addition it has been shown to
modulate BMP-6 expression by hepatic stellate cells in culture (28).
There is ample evidence that cytokines of this superfamily are present
in the adult liver and that TGF-
itself is an integral part of
disease processes in this organ. Further characterization of BMP-9
signaling in LECs and KCs downstream of receptor binding will enable a
more thorough understanding of the physiological utility of this
cytokine family in the context of liver function/dysfunction.
| |
ACKNOWLEDGEMENTS |
|---|
We thank L. F. Kolakowski, Ph.D. for help in analyzing the binding data and Mike DeBuysere for technical assistance.
| |
FOOTNOTES |
|---|
* This work was supported by National Institutes of Health Grant DK-19473.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.
§ To whom correspondence should be addressed: University of Texas Health Science Center at San Antonio, Dept. of Biochemistry, 7703 Floyd Curl Dr., San Antonio, TX 78284-7760. E-mail: olson@ biochem.uthscsa.edu; Tel.: 210-567-3770; Fax: 210-567-6595.
| |
ABBREVIATIONS |
|---|
The abbreviations used are: TGF, transforming growth factor; BMP, bone morphogenetic protein; KC, Kupffer cell; LEC, liver endothelial cell; PAGE, polyacrylamide gel electrophoresis; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; BS3, bis(sulfosuccinimidyl)- suberate.
| |
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