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INTRODUCTION |
Matrix GLA protein
(MGP)1 is a small ubiquitous
matrix protein containing carboxyglutamic acid (GLA) (calculated
mass of mature protein 10.4 kDa), initially isolated from bone
and characterized by Price et al. (1). Results from other
investigators suggest that MGP affects differentiation in developing
cartilage and bone. Luo et al. (2) observed that MGP is
expressed in early and late stages of chondrogenic differentiation but
not in the intermediate stage. Yagami et al. (3) found that
MGP had an effect on mineralization in chondrocytes that was dependent
on cell stage; it affected mineralization in hypertrophic chondrocytes
but not in proliferative chondrocytes. They also found that
overexpression of MGP in developing limb buds delayed chondrocyte
maturation and blocked endochondral ossification. In MGP null
(MGP
/
) mice (4), the epiphyseal growth plates of bones showed
inappropriate calcification in the layer of proliferating chondrocytes
that failed to differentiate into hypertrophic chondrocytes. In
addition, the mice unexpectedly developed severe vascular
calcification, resulting from a replacement of the aortic medial layer
by chondrocyte-like cells, producing a typical cartilage matrix that
progressively calcified.
When Urist and colleagues (5) first discovered bone morphogenetic
protein (BMP), they observed a tight association with MGP in
vitro during protein purification requiring strong denaturants to
break. Although complex formation was not shown in vivo or in situ, it was suggested that MGP may sequester BMP in bone
tissue and as such regulate its activity (5). Since BMP mediates its biological response through a cell surface receptor, MGP could potentially act extracellularly to form an inactive complex with BMP.
It has not been shown whether MPG affects BMP function by forming such
a biologically inactive complex whose formation blocks BMP action on
cells. In this paper, we test the hypothesis that the mechanism for the
effect of MGP on cell differentiation is through modulation of BMP activity.
A useful model for assaying BMP-2 functional activity in mesenchymal
differentiation is the "undifferentiated" mesenchymal cell line,
C3H10T1/2. These multipotent mouse embryonic cells, also known as
vascular precursor cells (6), differentiate along adipogenic,
osteogenic, and chondrogenic lineages when stimulated with BMP-2 (7,
8). Once stimulated, colonies of lineage form within the same culture
and time frame.
To test the hypothesis that MGP modulates BMP-2-induced mesenchymal
differentiation, we overexpressed human MGP (hMGP) and antisense to
hMGP (AS-hMGP), and we assayed differentiation along each lineage in
response to recombinant human BMP-2 (rhBMP-2). We also compared
responses to rhBMP-2 in cells isolated from aortas of homozygous and
heterozygous MGP-deficient mice to those isolated from aortas of
wild-type mice. FLAG-tagged hMGP with the same biological effect as
native hMGP was used to localize the MGP effect to the extracellular
space. In addition, the effect of overexpression of selected hMGP
subdomains was compared with that of full-length hMGP.
These results support the concept that MGP modulates BMP activity in
mesenchymal differentiation and that the physical interaction observed
during protein purification from bone may have physiological significance.
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MATERIALS AND METHODS |
Vector Construction--
Full-length human MGP (hMGP) cDNA
subcloned into the EcoRI site of the pBSSK(
) plasmid
(Stratagene, La Jolla, CA) was obtained from the American Tissue
Culture Collection (Manassas, VA) (9). The hMGP sequence was excised
and subcloned into pcDNA3.1(+) Zeo (Invitrogen, Carlsbad, CA) in
sense or antisense orientation using the HindIII and
XbaI sites for sense orientation, and the NotI and ApaI sites for antisense orientation. A schematic
overview of the vectors used in this study is provided in Fig.
1.

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Fig. 1.
Schematic overview of human MGP vectors.
The full-length hMGP and selected subdomains were inserted into the
pcDNA3.1(+) plasmid as described under "Materials and Methods."
N, N terminus; C, C terminus; P,
phosphorylation site; C-C, cystine bridge; ,
-carboxylation site.
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To construct the N-MGP vector (containing the N-terminal and the
central region of hMGP, aa 1-54, Fig. 1), the correct fragment was
generated by PCR of hMGP cDNA using primers ERIFL-5' and MGPX-3' (Table I) and then subcloned into the
EcoRI and XbaI sites in the pcDNA3.1(+)
plasmid after digestion of the restriction enzyme sites in the primers.
To construct the mid-MGP vector (containing the central region of hMGP
alone, aa 35-54, Fig. 1), the C-MGP vector (containing the C-terminal
and the central region of hMGP, aa 35-84, Fig. 1), and the control
vector (containing only the leader sequence, Fig. 1), the hMGP-leader
sequence was first excised from the hMGP cDNA using
EcoRI and a HinfI site located just downstream of
the leader sequence. Fragments containing the C-MGP and the mid-MGP
regions were generated by PCR of hMGP cDNA using primers MGPH-5'
and XBAFL-3', and MGPH-5' and MGPX-3' (Table I) respectively. The
fragments were subsequently ligated into the pcDNA3.1(+) vector downstream of and in frame with the leader sequence, after digestion of
the restriction enzyme sites in the PCR primers. For the control vector, the EcoRI-HinfI fragment was ligated into
pcDNA3.1(+) digested with EcoRI and EcoRV.
This vector contains the leader sequence alone followed by a stop codon
1 base downstream of the HinfI site.
To construct the FLAG-tagged hMGP vector, a fragment containing
full-length hMGP in which the stop codon had been replaced by an
XhoI site, was generated by PCR of hMGP cDNA using
primers ERIFL-5' and mgpFLAG-3' (Table I). The FLAG tag was placed in the C terminus of the resulting protein by subcloning the fragment into
the EcoRI and XhoI sites in the pCMV-Tag4 plasmid
(Stratagene), after digestion of the restriction enzyme sites in the primers.
To construct the human BMP-2 vector, the correct fragment containing
the complete coding sequence was generated by PCR of human BMP-2
cDNA using primers bmp2ERI-5' and bmp2XBA-3' (Table I), and then
subcloned into the EcoRI and XbaI sites in the
pcDNA3.1(+) plasmid, after digestion of the restriction enzyme
sites in the primers.
All constructs were confirmed by restriction enzyme analysis and DNA
sequencing of the complete coding sequences.
Cell Culture--
C3H10T1/2 mouse cells were cultured in
-minimum Eagle's medium with Earle's salt (Irvine Scientific,
Santa Ana, CA) and HEK293 cells in Dulbecco's modified Eagle's medium
(Irvine Scientific), both supplemented with 10% heat-inactivated fetal
bovine serum (FBS) (HyClone Laboratories, Logan, UT), penicillin (100 units/ml), streptomycin (100 units/ml), sodium pyruvate (1 mmol/L), and
L-glutamine (2 mM).
To prepare aortic cells from MGP-deficient and control mice, whole
aortas were obtained and cleaned of visible surrounding fat and other
tissue. Aortic cells from heterozygous MGP+/
and normal mice were
prepared by enzymatic digestions with cold trypsin as described
previously (10). Because of cartilaginous metaplasia, homozygous
MGP
/
aortas required use of enzymatic digestion adapted to
cartilage as described previously (10) for release of cells. After
digestion, the cells were plated in gelatin-coated 12-well plates and
grown in F-12 medium (Irvine Scientific) supplemented with 20%
heat-inactivated FBS, penicillin (100 units/ml), streptomycin (100 units/ml), sodium pyruvate (1 mmol/liter), and L-glutamine (2 mM). The MGP
/
cells did not tolerate subsequent
trypsinization and re-plating and thus were treated with rhBMP-2 as
they reached ~75% confluency. Cells from normal mice and MGP+/
mice were trypsinized, plated, and treated with rhBMP-2 as they reached
~75% confluency. Treatment with rhBMP-2 was continued for a total of
14-16 days. The cells were then taken to histochemical staining,
RT-PCR, or immunoblotting.
Transfections were performed using SuperfectTM (Qiagen,
Chatsworth, CA), after optimization of the ratio of DNA to transfection agent as per manufacturer's instructions. Stable, mixed mass
transfectants of C3H10T1/2 cells were selected and maintained by adding
ZeocinTM (Invitrogen) at a concentration of 500 ng/ml to
all culture media.
Recombinant human BMP-2 was graciously supplied by Genetics Institute
or generated in our laboratory from HEK293 cells transiently transfected with the human BMP-2 vector. The concentration of rhBMP-2
in undiluted conditioned media was estimated by comparison with
purified rhBMP-2 from Genetics Institute using Western blotting.
Differentiation Assay, C3H10T1/2 Cells--
Two thousand
C3H10T1/2 cells were plated in 60-mm Petri dishes, or 150 cells were
plated per well in 12-well plates. BMP-2 was added to the cultures the
day after plating at a concentration of 500 ng/ml. Because of the
sparse plating, the initial medium with rhBMP-2 was left on the cells
for 7 days; thereafter, the medium was changed every 3-4 days for a
total of 21 days. Cells in 60-mm Petri dishes were used for RT-PCR,
immunoblotting, or immunoradiometric assay; cells in 12-well plates
were used for histochemical staining.
RT-PCR--
Total RNA was isolated from cultured cells using an
RNA isolation kit (Stratagene). Reverse transcription of 3 µg of
total RNA was carried out for 90 min at 37 °C in 50 µl of RT
buffer (as supplied by Stratagene), supplemented with 0.5 mM of each dNTP (Amersham Pharmacia Biotech), 50-80 units
of RNase Block, 50 units of Moloney murine leukemia virus
reverse transcriptase (both Stratagene), and 750 ng of oligo(dT) (10)
(Roche Molecular Biochemicals) for priming.
PCR using primers to unique sequences in each cDNA was carried out
in a volume of 10 µl, using Pfu buffer, 7 units of native Pfu polymerase (Stratagene), 0.1 µl of
[
-32P]dCTP (Amersham Pharmacia Biotech), 25 ng of each
primer, and 1 µl of template (from a 50-µl RT reaction). Thermal
cycling was performed as follows: 1) initial denaturation at 96 °C
for 2 min; 2) cycling for cDNA-specific number of cycles (Table I)
between 96 °C for 1 min, cDNA-specific annealing temperature
(Table I) for 2 min, and 72 °C for 2 min; and 3) final extension at
72 °C for 5 min.
For semi-quantitative PCR, the number of cycles was chosen so that
amplification remained well within the linear range, as assessed by
densitometry (NIH Image J, version 1.08i, public domain program)
of standard curves for representative samples. An equal volume from
each PCR was analyzed by 6% nondenaturing polyacrylamide gel
electrophoresis, and dried gels were examined by autoradiography. For
PCR generating DNA fragments for vector construction, the number of
cycles was chosen to ensure sufficient amounts of generated product,
usually 45 cycles.
Alkaline Phosphatase (AP) Assay--
Cells were
plated in triplicate in 96-well plates at 100 cells per well. Sixteen
hours later, 200 µl of conditioned media containing rhBMP-2
(estimated 600 ng/ml) alone or in combination with ~3-fold molar
excess hMGP-FLAG was applied to the cultures. The initial medium was
left on the cells for 6 days; thereafter, the medium was changed every
3-4 days for a total of 13-14 days.
Conditioned media containing either BMP-2 or hMGP-FLAG alone were
prepared from HEK293 cells transfected with 16 µg of the respective
plasmid per 100-mm culture dish. Conditioned media containing both
BMP-2 and hMGP-FLAG were prepared from HEK293 cells transfected with 4 and 12 µg of the BMP-2 and the hMGP-FLAG plasmid, respectively, per
100-mm culture dish. In all cases, 5 ml of conditioned medium was
collected after 48 h of incubation starting after transfection.
Control media were collected form HEK293 cells transfected with empty vector.
To determine AP activity, the cells were washed once with
phosphate-buffered saline and lysed by freeze-thawing twice in 50 µl
of 0.2% Nonidet P-40 with 1 mM MgCl2. Cell
lysates were assayed for AP activity by adding 150 µl of AP buffer
(40 mg of Sigma 104 phosphate substrate dissolved in 20 ml of Sigma 221 Alkaline Buffer Solution diluted 1:2 with distilled water) per well and incubating for 60 min at 37 °C. Absorbance at 405 nm was read using
a microplate reader (Molecular Devices, Sunnyvale, CA), and activity
was expressed as units per mg of cellular protein.
Histochemical Staining--
Oil Red O, von Kossa, Alizarin Red,
and Alcian Blue histochemical stains were performed using
standard methods. Alkaline phosphatase stain was performed as described
previously (11).
Immunoblotting--
For immunoblotting, 30 µg per sample of
whole cell extract protein was electrophoresed through 3-8%
NuPAGETM Tris acetate gels (NOVEX, San Diego, CA) or
4-12% NuPAGETM Bis-Tris (MOPS) gels for collagen IX and
peroxisome proliferator-activated receptor
(PPAR-
),
respectively. For detection of FLAG-tagged hMGP and BMP-2 in
conditioned media, 30 µl of media were electrophoresed through 10%
NuPAGETM Bis-Tris (MES) gels. Proteins were transferred to
nitrocellulose filter using NuPAGETM Transfer buffer
(NOVEX). The blots were incubated with specific antibodies to either
collagen IX (monoclonal antibody MAB3304 (12), Chemicon International,
Temecula, CA) at a concentration of 2.5 µg/ml, PPAR-
protein
(polyclonal antibody H-100, Santa Cruz Biotechnology, Santa Cruz, CA)
at a concentration of 1 µg/ml, the FLAG tag (polyclonal antibody
F7425, Sigma) at a concentration of 1 µg/ml, or BMP-2 (polyclonal
antibody H-51, Santa Cruz Biotechnology) at a concentration of 1 µg/ml. Specific antibody binding was visualized with the
appropriate horseradish peroxidase-conjugated anti-IgG secondary
antibody (Santa Cruz Biotechnology).
Immunoradiometric Assay--
Mouse osteocalcin was quantitated
in undiluted conditioned culture medium after 4 days of incubation
according to manufacturer's instructions using an immunoradiometric
assay (Immutopics, San Clemente, CA), and the results were expressed as
ng/ml. There was no cross-reactivity with bovine osteocalcin derived
from FBS.
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RESULTS |
Transfection of Sense and Antisense Human MGP in C3H10T1/2
Mesenchymal Cells--
To determine the effect of MGP on
BMP-2-directed differentiation, we used low density cultures of the
multipotent mouse mesenchymal cell line, C3H10T1/2 cells, which commit
to various mesenchymal lineages when stimulated with exogenous BMP-2
(7, 8), even after brief, transient exposures (7). Without stimulation, there are low levels of spontaneous differentiation at base line (7).
We used these cells as a bioassay to study effects of MGP on BMP-2
response by varying MGP expression.
MGP expression is highly dependent on cell density in cultured rat
kidney (NRK) cells (13); the expression at low density was barely
detectable but increased significantly with culture density. We
observed the same phenomenon in C3H10T1/2 cells, with barely detectable
MGP expression 1 day after low density plating, increasing at least
12-13-fold after 3 weeks in culture.
To test the hypothesis that MGP affects cell differentiation through
altered responses to BMP-2, we modulated the MGP expression at low cell
density (i.e. minimal expression of endogenous MGP) conditions, using stable cell lines overexpressing human MGP (hMGP) or
antisense to MGP (AS-hMGP). We used the human MGP sequence that can be
distinguished from mouse MGP using RT-PCR.
One day after low density plating, cells transfected with hMGP had an
estimated 3-fold increase in MGP signal (including both sense mouse and
sense human MGP, Fig. 2). Also at low
density, cells transfected with AS-hMGP had an estimated 3-fold
increase in MGP signal (including both sense mouse and
antisense human MGP, Fig. 2) compared with nontransfected
control cells. Addition of BMP-2 in all experiments was performed at
this low density state.

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Fig. 2.
Relative expression at low density of hMGP
(sense) and AS-hMGP (antisense) in
C3H10T1/2 cells transfected with hMGP or AS-hMGP, respectively,
compared with expression of mouse MGP in nontransfected
(NT) control cells. The relative expression was
determined by multiple cycles of RT-PCR using primers recognizing
either both mouse and human MGP or human MGP alone. Multiple
radiographs were scanned, and the values were normalized to GAPDH and
expressed relative to the MGP expression of subconfluent NT control
cells (1-fold). The results are shown as the mean and S.D. of at least
three independent determinations.
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Effect of MGP Overexpression on BMP-induced
Differentiation--
To assess whether altered levels of MGP
expression modulate BMP-2-induced differentiation, we established a
differentiation assay in C3H10T1/2 cells following the methods of Wang
et al. (7). Treatment with rhBMP-2 or control vehicle was
started at low density and continued for 21 days to allow distinct
colonies of differentiated cells to form. We used rhBMP-2 at a
concentration of 500 ng/ml based on preliminary experiments showing
that this concentration induced differentiation in nontransfected
C3H10T1/2 cells detectable by both specific cell markers and
histochemical stains used to identify osteogenic, chondrogenic, and
adipogenic cell differentiation. RT-PCRs for osteocalcin (14) in
combination with von Kossa and Alizarin Red stains were used to
identify osteogenic differentiation, RT-PCR for collagen IX (15), and
Alcian Blue stain to identify chondrogenic differentiation, RT-PCR for
PPAR-
(16), and Oil Red O stain to identify adipogenic differentiation.
The response to BMP-2 differed with the transfected construct.
Nontransfected cells at low density underwent differentiation into a
mix of all three cell lineages with cells of each lineage grouped in
colonies as expected. No difference was seen between nontransfected
cells and control cells transfected with plasmid containing only the
MGP-leader sequence. However, in cultures of cells overexpressing hMGP,
BMP-2-induced differentiation was inhibited. Conversely, in cultures
overexpressing AS-hMGP, osteogenesis and chondrogenesis were enhanced.
These effects were first evident in phenotypic changes. Cells
overexpressing hMGP maintained a noninduced phenotype despite BMP-2
treatment (Fig. 3, middle
panels). In contrast, even without BMP-2 treatment,
AS-hMGP-expressing cells spontaneously underwent these phenotypic
changes (Fig. 3, bottom panels).

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Fig. 3.
Morphology of C3H10T1/2 cells transfected
with hMPG and AS-hMGP after 14 days in culture with treatment with
vehicle alone (left panels) or rhBMP-2 (500 ng/ml)
(right panels). Nontransfected (NT)
control cells are shown for comparison. Original magnification × 40.
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In hMGP-overexpressing cells, cell-specific markers, osteocalcin and
collagen IX, were inhibited based on semi-quantitative RT-PCR (Fig.
4). In fact, both osteocalcin and
collagen IX expression showed a decreasing trend in BMP-2-treated
cells. In contrast, in AS-hMGP-overexpressing cells, osteocalcin
expression increased about 2-fold, and collagen IX expression increased
4-fold compared with non- and control-transfected cells.

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Fig. 4.
Effect of varying MGP expression on marker
gene expression in response to BMP-2. Upper panel,
expression of differentiation markers in C3H10T1/2 cells transfected
with hMGP or AS-hMGP after 21 days in culture; treatment with vehicle
alone ( ) or rhBMP-2 (500 ng/ml) (+), compared with nontransfected
(NT) and control-transfected (CT) cells. Total
RNA and cDNA was prepared, and PCR was performed using specific
primers for osteocalcin (osteogenic differentiation), collagen IX
(chondrogenic differentiation), and PPAR- 2 (adipogenic
differentiation). GAPDH is shown for comparison. Lower
panel, relative signal intensity of the three markers after
normalization to GAPDH. The results are expressed as fold increase of
expression with respect to vehicle-treated control and are shown as the
mean and S.D. of at least three independent determinations.
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Adipogenic differentiation appeared to be only partially
inhibited in cells expressing hMGP based on only partial inhibition of
PPAR-
2. This may be due to the base-line expression of BMP-4 which
induces adipogenic differentiation in these cells (17).
To confirm the RT-PCR results, we performed immunoradiometric
quantitation of mouse osteocalcin in cell media (Fig.
5, upper panel) and
immunoblotting for PPAR-
protein and collagen IX (Fig. 5,
lower panel). These results were consistent with the
findings of the RT-PCR. The antibody for PPAR-
protein recognizes
both PPAR-
1, which is widely expressed in a variety of cells (16), and PPAR-
2. PPAR-
2 contains 28 extra amino acids at the N
terminus (16) (~3.5 kDa) and thus corresponds to the upper band.

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Fig. 5.
Effect of varying expression on marker
protein levels in response to BMP-2. Mouse osteocalcin in media as
determined by immunoradiometric assay (top), collagen IX
(middle), and PPAR- protein (bottom) in cell
extracts as shown by immunoblotting from C3H10T1/2 cells transfected
with hMGP or AS-hMGP after 21 days in culture: treatment with vehicle
alone ( ) or rhBMP-2 (500 ng/ml) (+), compared with nontransfected
(NT) and control-transfected (CT) cells. 30 µg
of protein was loaded per lane for immunoblotting. Fold induction was
determined by comparison of the signal intensity of treated cells to
that of nontreated cells.
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Nonquantitative histochemical stainings (Fig.
6) further confirmed an inhibition of
osteogenic and chondrogenic differentiation in hMGP-overexpressing
cells. As expected, Oil Red O staining showed occasional adipocytes
(Fig. 6, left middle panel). Positive staining for all
lineages was seen in nontransfected control cells and in
AS-hMGP-overexpressing cells.

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Fig. 6.
Histochemical staining of C3H10T1/2 cells
transfected with hMGP and AS-hMGP after treatment with rhBMP-2 for 21 days. Nontransfected (NT) control cells are shown in
the upper panels, hMGP cells in the middle
panels, and AS-hMGP cells in the lower panels. Oil Red
O staining (adipogenic differentiation) is shown in the left
panels, von Kossa staining (osteogenic differentiation) in the
middle panels, and Alcian Blue staining (chondrogenic
differentiation) in the right panels. Original
magnification × 40.
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Thus, in cells overexpressing hMGP, BMP-2 activity was reduced, and in
cells overexpressing AS-hMGP, BMP-2 activity was increased.
Effect of BMP-2 on MGP-deficient Mouse Cells--
To test our
results ex vivo, mixed aortic cells from homozygous MGP
/
and heterozygous MGP+/
mice as well as wild-type controls were
harvested and cultured. The cells were treated with rhBMP-2 at a
concentration of 500 ng/ml for 14-16 days.
When MGP
/
cells were treated with BMP-2, chondrogenic
differentiation was induced based on strong induction of alkaline phosphatase in colonies of rounded cells (Fig.
7A) and formation of distinct
colonies of chondrocytes producing acidic mucopolysaccharides (18)
(Fig. 7A). In MGP+/
or control +/+ cells, BMP-2 failed to
induce chondrogenic differentiation based on histochemical staining, as
well as RT-PCR and immunoblotting for collagen IX (data not shown).
However, in MGP+/
cells, BMP-2 did induce osteogenic differentiation
based on mineral formation and increased osteocalcin expression (Fig.
7, B and C). Although a low level of osteocalcin expression was found at base line in wild-type cells, this showed a
decreasing trend with BMP-2. The differential effect may be the result
of a changed ratio of MGP relative to BMP-2 resulting in relatively
unopposed BMP activity.

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Fig. 7.
A, histochemical staining for alkaline
phosphatase (top) and cartilage acidic mucopolysaccharides
(Alcian Blue) (bottom) demonstrating the effect of rhBMP-2
(500 ng/ml) for 14-16 days on mixed aortic cells isolated from
homozygous MGP null mouse (MGP / ). B, histochemical
staining for mineral (Alizarin Red) demonstrating the effect of rhBMP-2
for 14-16 days on aortic cells isolated from wild-type mouse (MGP+/+)
(left), and heterozygous MGP null mouse (MGP+/ )
(right). Original magnification × 100. C,
expression of osteocalcin in MGP+/+ and MGP+/ cells after treatment
with vehicle alone ( ) or rhBMP-2 (500 ng/ml) (+) for 14-16 days.
Fold induction was determined by comparison of the signal intensity for
treated cells to that of nontreated cells.
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These results differ from those in C3H10T1/2 cells, in that osteogenic
and chondrogenic differentiation in mouse cells appeared to be confined
to heterozygous or homozygous cells, respectively. It is possible that
the C3H10T1/2 cells are less differentiated than the isolated vascular
cells, retaining a higher degree of pluripotentiality. Equally possible
is that cell isolation procedures and/or culture conditions select for
certain types of cells resulting in the observed differences.
Effect of MGP-FLG on BMP-induced Differentiation--
In the
experiments described above, mRNA levels of MGP were modulated by
overexpression of hMGP or AS-hMGP or by using cells from MGP-deficient
mice. Even though mRNA levels most commonly reflect protein levels,
this may not always be the case. To determine directly the effect of
MGP protein on BMP-induced differentiation, we tagged the C-terminal
end of hMGP for easy detection in conditioned media (Fig.
8A, inset), and we compared
the response to BMP-2 of C3H10T1/2 cells expressing hMGP-FLAG with
those expressing nontagged hMGP. No significant difference was
detected, here demonstrated by comparing BMP-2-induced alkaline
phosphatase activity, an early marker for osteoblastic and chondrocytic
differentiation (Fig. 8A). These results indicate that MGP
protein is secreted by the transfected cells and that the
experimentally tagged protein retains the function of the original
MGP.

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Fig. 8.
A, alkaline phosphatase activity in
C3H10T1/2 cells transfected with hMGP and hMGP-FLAG after 12 days in
culture with treatment with vehicle alone or rhBMP-2 (500 ng/ml).
Nontransfected (NT) and control-transfected (CT)
cells are shown for comparison. Inset, Western blot
demonstrating secreted hGMP-FLAG in conditioned media. B,
alkaline phosphatase activity in nontransfected C3H10T1/2 cells after
12 days in culture with treatment with rhBMP-2 (estimated 500 ng/ml)
alone or in combination with ~3-fold molar excess hMGP-FLAG. Control
media and hMGP-FLAG alone are shown for comparison. Inset,
Western blot demonstrating secreted rhBMP-2 in conditioned media.
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Nontransfected C3H10T1/2 cells were then treated with conditioned media
containing BMP-2 alone or in combination with ~3-fold molar excess
hMGP-FLAG. Conditioned media containing BMP-2 (Fig. 8B,
inset) or hGMP-FLAG was prepared from HEK293 cells transfected with expression vectors of BMP-2 or hMGP-FLAG. Conditioned media containing both proteins was obtained by cotransfecting the
hMGP-FLAG and the BMP-2 vector at a 3:1 molar ratio or by combining
conditioned media from singly transfected cells at a comparable ratio.
Alkaline phosphatase was determined using a 96-well assay after 12-14
days of incubation with conditioned media (Fig. 8B). The
results show that exogenous addition of hMGP-FLAG to BMP-2-containing media inhibits BMP-2-induced differentiation as measured by alkaline phosphatase. No significant difference was seen in experiments using
conditioned media from cotransfected HEK293 cells versus singly transfected cells. This result places the site of action for MGP extracellularly.
Taken together, the three sets of experiments described above suggest
that MGP directs mesenchymal differentiation by modulating BMP activity.
Effects of MGP Domains on BMP-induced Differentiation--
To test
whether fragments of MGP would retain the effect of full-length MGP on
BMP-2-induced differentiation, we established stable cell lines
overexpressing selected subdomains of hMGP by transfection of C3H10T1/2
cells. Three vectors were transfected, one with the mid-region (usually
subjected to
-carboxylation) alone (mid-MGP, aa 35-54) and the
other two with the mid-region combined with either the N terminus
(N-MGP, aa 1-54) or the C terminus (C-MGP, aa 35-84) (Fig. 1). Unique
RT-PCR products for each vector were detected from cells transfected
with the respective vector using primers recognizing only human MGP
(Fig. 9, upper panel). The
relative expression by RT-PCR was determined by multiple cycles of
RT-PCR using comparable primer pairs and adjustment to GAPDH expression
and length of amplified sequence. Expression was compared with that of
hMGP-expressing cells (Fig. 9, lower panel). The lowest
expression was found in N-MGP cells, which is comparable to that of
confluent AS-hMGP. Intermediate expression was found in hMGP and
mid-MGP cells, and the highest expression is found in C-MGP cells.

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Fig. 9.
Top panel, RT-PCR demonstrating
specific expression of transfected constructs containing hMGP (sense
or antisense orientation) and selected subdomains of hMGP in C3H10T1/2
cells after 14 days in culture. The sense PCR primer for human MGP
(Table I) was used as the upstream primer for all constructs. As
downstream primers, the antisense human MGP-primer was used for the
hMGP, AS-hMGP, and the C-MGP constructs; the MGPX-3'-primer was used
for the N-MGP and the mid-MGP construct. The size of the respective PCR
product is shown to the left. Bottom panel,
relative signal intensity of the different constructs after
normalization to GAPDH and length of PCR product. The signal
corresponding to hMGP expression was regarded as 1-fold.
|
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The phenotype of cells expressing N-MGP or the mid-MGP resembled that
of cells expressing AS-hMGP (Fig. 4) whereas that of cells expressing
C-MGP did not. In N-MGP- and mid-MGP-expressing cells, there was a
significant enhancement in osteogenic differentiation as assessed by
RT-PCR of osteocalcin (Fig. 10). Even
without added BMP-2, osteogenic differentiation occurred in these cells
based on an approximate 4-fold increase in osteocalcin expression
compared with non- and control-transfected cells (Fig. 10) and positive mineral production (von Kossa staining, data not shown). Addition of
BMP-2 had little additional effect on osteocalcin expression in N-MGP
cells but induced it about 2-fold in mid-MGP cells (Fig. 10), which may
be attributable to the higher relative expression of this construct
compared with N-MGP (Fig. 8) or to specific characteristics of this MGP
domain. Chondrogenic differentiation was also enhanced about 2-fold in
N-MGP cells and 4-fold in mid-MGP cells compared with non- or
control-transfected cells as assessed by RT-PCR for collagen IX (Fig.
10). This finding suggests that the mid-MGP fragment is the most
efficient in enhancing BMP-2 activity either due to its particular
level of expression or domain characteristics.

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Fig. 10.
Upper panel, expression of
cell-specific differentiation markers in cells transfected with
selected domains of hMGP after 21 days of culture: treatment with
vehicle alone ( ) or rhBMP-2 (500 ng/ml) (+), compared with
nontransfected (NT) and control-transfected (CT)
cells. Total RNA and cDNA were prepared, and PCR was performed
using specific primers for osteocalcin (osteogenic differentiation) and
collagen IX (chondrogenic differentiation). GAPDH is shown for
comparison. Lower panel, relative signal intensity of the
two markers after normalization to GAPDH. The results are expressed as
fold increase of expression in respective vehicle-treated control and
are the means of at least three independent determinations.
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In cells expressing C-MGP, no enhancement of osteocalcin was seen at
base line, and the response to BMP-2 overall was not different from
that of control cells (Fig. 10). Induction of adipogenic differentiation was similar in all cells, between 2.5- and 3.5-fold, without clear enhancement or inhibition (data not shown).
Thus, the intact MGP protein appears to be required to inhibit BMP-2
activity, whereas the N-MGP and mid-MGP subdomains have the opposite
effect. One possible mechanism is through interference with MGP function.
 |
DISCUSSION |
A possible clue to the mechanism of the effect of MGP on
differentiation comes from earlier reports that MGP is associated with
BMP, during BMP purification from bone (5). To determine whether the
observed association has a functional significance, and whether it
serves as the mechanism for MGP's modulation of differentiation, we
tested the effect of MGP on BMP activity. The findings presented in
this paper suggest that MGP modulates BMP-2-induced differentiation.
In C3H10T1/2 cells, MGP overexpression suppresses the effects of BMP-2,
whereas overexpression of antisense to MGP enhances these effects. It
is not clear how AS-hMGP exerts its effect. The most straightforward
explanation is that the 3-fold excess of AS-hMGP mRNA (Fig. 2)
reacts with the endogenous MGP mRNA to decrease its translation.
Our results show that even when present at only 3-fold excess in the
media, hMGP-FLAG inhibits the response to BMP-2, which suggests that
small changes in MGP expression and MGP levels may have significant
impact. Alternatively, inhibitory peptides may be produced from the
reversed hMGP sequence. Inspecting the sequence, translation of two
potential peptides (22 and 6 amino acids respectively) is theoretically
possible. However, extensive GenBankTM searches did not
show any obvious similarity with known proteins, including MGP and BMP,
which could explain the effect of AS-hMGP.
The importance of low density plating of C3H10T1/2 cells to be able to
study the differentiation of individual cells or colonies and to avoid
confusing results due to too high cell density has been emphasized by
Wang et al. (7). Interestingly, there appears to be a lower
cell density in cultures that failed to differentiate. Since the cells
were plated carefully at identical densities, this difference would be
attributed to BMP-2 treatment. Two possibilities are that BMP-2
directly affects cell density or that induction of differentiation by
BMP-2 affects cell density. The latter is more likely, based on the
results of Wang et al. (7) that induction of differentiation
by BMP-2 causes a decrease in contact inhibition resulting in
ultimately greater cell density. Thus, the apparent difference in cell
density is an expected result, rather than a cause of the differentiation.
All markers of osteogenic differentiation increased in BMP-2-treated
AS-hMGP cells except for one, osteocalcin levels measured in the media.
This phenomenon of decreased secretion of soluble osteocalcin in
BMP-2-stimulated cells undergoing osteogenic differentiation has been
described previously (19) and may be attributed to adsorption of
osteocalcin by hydroxyapatite mineral.
Although osteogenic and chondrogenic differentiation was affected, the
effect of MGP on adipogenic differentiation was variable. Even though
C3H10T1/2 cells express minimal amounts of BMP-2 at base line (20),
they do express other BMP (20, 21) including significant amounts of
BMP-4, a strong adipogenic inducer in these cells (17). Thus, other
factors regulating adipogenesis may explain the variable effect on adipogenesis.
Ex vivo results showed that BMP-2 induced chondrogenic and
osteogenic differentiation in MGP
/
and MGP+/
cells, respectively, which differs from the results in the C3H10T1/2 cells. In C3H10T1/2 cells, intermediate levels of BMP-2 have been reported to favor formation of osteoblastic cells, and high levels to favor chondrocytes (7), but not to the exclusion of differentiation along other lineages.
It is possible that the procedures used to obtain cells from the
respective aortas may select for cells with more limited inducibility
compared with C3H10T1/2 cells. Culture conditions and time in culture
may be other factors affecting the observed differentiation.
MGP appears to be an important factor in ensuring correct
differentiation of vascular smooth muscle cells based on the profound changes seen in MGP-deficient mice where the whole media are replaced by chondrocyte-like cells undergoing endochondral ossification (4).
Interestingly, BMP-2 is expressed in the embryonic aorta (embryonic day
10.5) when the aorta is still a tube lined with a single layer of cells
(22), coinciding with the time of initial media formation (23).
In vitro experiments have shown that BMP-2 is able to induce
smooth muscle cell differentiation in neural crest-derived stem cells
(22). We speculate that the absence of MGP may allow for uncontrolled
BMP-2 activity at a stage when smooth muscle cell precursors are easily
triggered to undergo endochondral bone formation leading to the
phenotype seen in MGP-deficient mice.
By FLAG-tagging hMGP and using HEK293 cells for producing MGP, we
facilitated both the detection of the protein in medium and avoided
handling of purified hMGP which is poorly soluble in water-based
buffers (1). Functionally, hMGP-FLAG retained the function of untagged
hMGP when expressed in C3H10T1/2 cells. The same effect was seen when
hMGP-FLAG was added to the culture medium that localizes the action of
MGP to the extracellular space. A possible in vivo scenario
is that MGP binds BMP-2, preventing its interaction with cell surface
receptors and retaining BMP during integration with bone matrix. This
mechanism would account for the earlier observation of BMP being
tightly associated with MGP during purification of bone proteins
(5).
The effect of full-length MGP did not seem to be retained by any of the
three selected subdomains. Instead, two of the three subdomains had the
reverse effect of full-length MGP, may be due to different expression
levels or domain characteristics. MGP is mainly attached to the organic
matrix if bone (24). Thus, possible mechanisms for the MGP fragments
would be to interfere with the matrix attachment of MGP or its
interaction with BMP, in that way enhancing BMP activity.
Alternatively, the MGP domains may activate other or additional
osteoinductive factors such as GDF-7 (21), possibly explaining the
base-line differentiation observed without BMP-2 treatment in N-MGP-
and mid-MGP-expressing cells.
Further support for the role of MGP in cell differentiation is that MGP
expression is strictly limited to specific stages and zones of
chondrocyte development (2) and that the MGP null mouse has
disorganized epiphyseal growth plates (4). In addition, Yagami et
al. (3) showed that overexpression of MGP in developing limb buds
delayed chondrocyte maturation and blocked endochondral ossification.
There are precedents for modulation of BMP activity. Embryonic
regulators including chordin (25), noggin (26), gremlin, cerberus, and
DAN (27) directly bind and inhibit BMP despite their widely different
primary structures. In the case of transforming growth factor-
, a
member of the same superfamily of growth factors as BMP, the matrix
protein betaglycan sequesters transforming growth factor-
and limits
access to signaling receptors (28, 29).
Together, our findings suggest MGP modulates mesenchymal cell
differentiation and that the mechanism is, at least in part, modulation
of the potent bone differentiation factor, BMP-2.