From the Division of Bone and Mineral
Diseases, § Renal Division, and ¶ Cardiovascular
Division, Department of Internal Medicine, Washington University
School of Medicine, St. Louis, Missouri 63110
Received for publication, November 3, 2000, and in revised form, January 25, 2001
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ABSTRACT |
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Extracellular signal-regulated kinases
(Erks), members of the mitogen-activated protein kinase superfamily,
play an important role in cell proliferation and differentiation. In
this study we employed a dominant negative approach to determine the
role of Erks in the regulation of human osteoblastic cell function. Human osteoblastic cells were transduced with a pseudotyped retrovirus encoding either a mutated Erk1 protein with a dominant negative action
against both Erk1 and Erk2 (Erk1DN cells) or the LacZ protein (LacZ
cells) as a control. Both basal and growth factor-stimulated MAPK
activity and cell proliferation were inhibited in Erk1DN cells.
Expression of Erk1DN protein suppressed both osteoblast differentiation
and matrix mineralization by decreasing alkaline phosphatase activity
and the deposition of bone matrix proteins. Cell adhesion to collagen,
osteopontin, and vitronectin was decreased in Erk1DN cells as compared
with LacZ cells. Cell spreading and migration on these matrices were
also inhibited. In Erk1DN cells, expression of
For bone formation to occur, osteoblast precursor cells must
migrate from bone marrow compartment to bone surface where they differentiate and deposit bone matrix. Several growth factors including
FGF-2,1 TGF- Numerous signal transduction pathways are induced by integrin-matrix
interactions in a variety of cell systems (6-10). The Ras/MAPK signal
transduction pathway is one of the best characterized pathways.
Adhesion of cells to collagen, fibronectin, or vitronectin has been
shown to induce MAPK activity in many cell systems (24, 25). Growth
factors have also been shown to activate MAPK activity (26, 27).
Although both integrins and growth factors propagate their signals via
MAPK, the induction of MAPK by integrin-matrix interaction is prolonged
whereas growth factor-stimulated MAPK is more transient and requires
integrin-mediated cell adhesion (24, 28, 29). Conversely, growth
factors can regulate integrin expression (30, 31). Thus, MAPK mediates
two important signal transduction pathways, one initiated by integrins
and the other by growth factors, which cross-talk to each other and
further emphasize the importance of MAPK in regulating a variety of
cell functions.
It has been documented that MAPK activity is stimulated in osteoblasts
when they adhere to type I collagen (32). However, no data are
currently available with regard to the MAPK activity when osteoblasts
adhere to other bone matrix proteins. We have previously shown that
MAPK is activated by growth factors in osteoblasts (27). Since
integrin-matrix interaction and growth factors regulate osteoblast
growth and differentiation, MAPK may play an important role in
mediating these osteoblastic activities in bone formation. Therefore,
in this study, we analyzed the role of Erks of the MAPK superfamily in
the regulation of osteoblast growth and differentiation by
overexpressing a dominant negative Erk1 (Erk1DN) protein in these
cells. We also examined the effects of Erk1DN protein on the expression
of integrins and the migration, adhesion, and spreading properties of osteoblasts.
Materials--
Type I collagen (Vitrogen 100) was from Cohesion
(Palo Alto, CA). Recombinant osteopontin was purified as described
previously (33). [125I]NaI and Tran35S-Label
were from ICN (Costa Mesa, CA). [3H]Thymidine,
[ Cell Cultures--
Human osteoblasts and bone marrow stromal
cells were isolated from ribs as described previously (33, 34).
Briefly, the ribs were cleaned of any extraneous tissue and then split.
The trabecular bone chips were recovered by bone curette and were subjected to a 2-h collagenase (1 mg/ml) digestion. After digestion, bone chips were plated into culture flasks. Osteoblasts outgrown from bone chips were subcultured. Bone marrow stromal cells, which represent relatively immature preosteoblastic cells, were also isolated
from bone marrow using Histopaque-1077 gradient followed by adhesion to
culture flasks. Both osteoblasts and bone marrow stromal cells were
subcultured in Generation of Pseudotyped Retrovirus Carrying a Dominant Negative
Erk1 cDNA--
A kinase-defective Erk1 cDNA (35, 36), in which
the AA nucleotides at 211 and 212 positions were mutated to CG,
resulting in replacing amino acid Lys-71 with Arg (Erk1 K71R), was
kindly provided by Dr. Melanie H. Cobb (University of Texas,
Southwestern Medical Center, Dallas, TX). Erk1 K71R has been shown to
function in a dominant negative fashion and can block both Erk1 and
Erk2 activities (37). The Erk1 K71R cDNA was cloned into the
NcoI and BamHI sites of SFG retroviral vector as
described previously (38, 39). For generation of retroviral particles
pseudotyped with vesicular stomatitis virus G glycoprotein, SFG-Erk1
K71R viral vector was transfected using LipofectAMINE into the 293GPG packaging cell line, which expresses the MuLV gag-pol, and also expresses the vesicular stomatitis virus G glycoprotein under tetracycline regulation (38, 39). The conditioned medium was harvested
daily after the withdrawal of tetracycline, and the media harvested
from day 3 to day 7 containing the highest titer of viral particles
( Transduction of Human Osteoblastic Cells with Pseudotyped
Retroviruses--
The day before transduction, HOB cells were seeded
in 150-mm culture dishes (1 × 106/dish). The next
day, 25 ml of conditioned medium containing Erk1DN or LacZ virus was
added to each dish, and the incubation continued for another 24 h.
At the end of the incubation period, the virus medium was removed, and
cells were grown to confluence in Immunostaining of the Transduced Cells for Erk Proteins--
HOB
cells were seeded onto coverslips and allowed to recover overnight.
Cells were then transduced with either LacZ or Erk1DN virus for 24 h, followed by further incubation in the growth medium for an
additional 48 h. Cells were washed twice with phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde in PBS for 15 min. After
washing twice with PBS, cells were treated with ammonium chloride to
quench any remaining paraformaldehyde. Cells were again washed with PBS
and then permeabilized with 0.1% Triton X-100 in PBS (PT), followed by
incubation with anti-pan-Erk antibody (1:400) in PT containing 1%
bovine serum albumin (BSA) for 1 h at room temperature. After
washing three times with PT, cells were incubated with Cy3-conjugated
goat anti-mouse antibody (1:200) in PT containing 1% BSA for 1 h.
Finally, after extensive washing with PT, the coverslips were mounted
on slides, and fluorescence was observed using an epifluorescence microscope.
MAPK Activity Assay--
Confluent cells in 100-mm culture
dishes were incubated in Western Blot Analysis--
Cell extracts (40 µg of protein),
isolated as described above, were subjected to SDS-PAGE and
transblotted to Immobilon-P membranes. The membranes were dried
completely and then incubated with the appropriate primary antibody
(1:1000) in PBS containing 1% casein and 0.04% Tween 20 for 1 h
at room temperature. After three PBS washes, the membranes were
incubated with a horseradish peroxidase-conjugated secondary antibody
(1:2000 in PBS containing 1% casein and 0.04% Tween 20) for 30 min
followed by another four PBS washes. Protein bands were visualized by
chemiluminescence using an ECL kit.
Nuclear Extract Preparation and Electrophoresis Mobility Shift
Assay (EMSA)--
Nuclear extracts were prepared as previously
described (31). Briefly, HOB cells were lysed in ice-cold buffer
containing 10 mM Hepes-KOH, pH 7.9, 1.5 mM
MgCl2, 10 mM KCl, 0.5 mM
dithiothreitol (DTT), 0.5 mM phenylmethylsulfonyl fluoride
(PMSF), and 0.6% Nonidet P-40. After centrifugation, nuclear pellets
were extracted with a high salt buffer (20 mM Hepes-KOH, pH
7.9, 1.2 mM MgCl2, 420 mM NaCl,
25% glycerol, 0.5 mM DTT, 0.5 mM PMSF, 10 µg/ml each leupeptin and pepstatin, and 25 µg/ml aprotinin) for
1 h. For EMSA, radioactive synthetic double-stranded AP-1
consensus oligonucleotide, labeled with T4 polynucleotide kinase and
[ RSK2 Phosphotransferase Activity Assay--
Immunocomplex kinase
assay was employed to measure the phosphotransferase activity of RSK2.
Cells were washed in ice-cold PBS three times and extracted with lysis
buffer consisting of PBS, pH 7.4, 1% Triton X-100, 1% Nonidet P-40,
0.5% sodium deoxycholate, 0.5 mM DTT, 1 mM
Na3VO4, 2 mM EGTA, 2 mM
EDTA, 5 mM Cell Proliferation Assay--
Viral-transduced HOB cells were
seeded in a 24-well plate (20,000 cells/well) and allowed to recover
overnight in Alkaline Phosphatase Activity Assay--
Confluent
viral-transduced cells in 24-well plates were washed three times with
Tris-buffered saline (20 mM Tris-HCl, pH 7.4, and 0.9%
NaCl), scraped into 0.5 ml of 50 mM Tris-HCl, pH 7.4, and
sonicated in a Fisher Dismembrator (30-40% of maximum
strength). Alkaline phosphatase activity in the sonicate was measured
as described previously (34).
Matrix Mineralization Assay--
Mineralized matrix was detected
by alizarin red-S staining for deposited calcium. Osteoblasts in
six-well culture plates were incubated for 28 days in mineralization
medium consisting of Cell Adhesion and Spreading Assays--
Adhesion to type I
collagen, osteopontin, or vitronectin was performed as described
previously (33). Briefly, single cell suspensions were obtained after
collagenase and trypsin/EDTA digestion. After rinsing with serum-free
Cell Migration Assay--
The day before the assay, Costar
transwell membranes were coated with collagen (100 ng), osteopontin
(250 ng), or vitronectin (250 ng) for 3 h at room temperature.
Single cell suspensions obtained as described above for the adhesion
assay were delivered into the transwells (100,000 cells in 0.1 ml/transwell) suspended inside the bottom wells containing 0.6 ml of
Cell Surface and Metabolic Labeling and Immunoprecipitation of
Integrins--
Cells in 150-mm culture dishes were surface-labeled
with [125I]NaI as described previously (33). After
labeling, cells were extracted with cell lysis buffer consisting of 10 mM Tris-HCl, pH 8.5, 0.15 M NaCl, 1 mM CaCl2, 0.02% NaN3, 2% Renex
30, and 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride
(AEBSF). Aliquots (~400 µl) of equal trichloroacetic acid-
precipitable radioactivity were incubated overnight with monoclonal
antibodies against Protein Measurement and Quantification of Band Intensity on X-ray
Films--
Protein concentration was measured by using the Bio-Rad
protein DC assay kit. To quantify the band intensity, x-ray films were
subjected to image analysis using ISS SepraScan 2001 (Integrated Separation Systems, Natick, MA).
Statistics--
Statistical analyses were performed using
Student's unpaired t test. Each experiment was performed at
least twice and the representative data were presented as mean ± S.E.
Erk/MAPK Activity Is Induced upon Adhesion of Human Osteoblastic
Cells to Bone Matrix Proteins--
HOB cells have been shown to adhere
to numerous bone matrix proteins, including type I collagen,
osteopontin, vitronectin, fibronectin, and bone sialoprotein, in an
integrin-dependent fashion (12, 15, 33). It has been well
established in various cell systems that Erk/MAPK activity is
stimulated by cell adhesion to matrix (6, 8-10). When HOB cells
adhered to type I collagen, fibronectin, osteopontin, or vitronectin,
both Erk-2 (major band at 42 kDa, the main Erk expressed in normal
human osteoblasts (Ref. 27)) and Erk-1 (minor band at 44 kDa)
activities were consistently enhanced (4.9-7.4-fold after
normalization with the corresponding tubulin protein level) as
determined by Western blot analysis using antibody against the active
phosphorylated Erks (p-Erk) (Fig. 1).
Likewise, the total MAPK enzymatic activity, determined by the
incorporation of 32P into PHAS-1 substrate, was stimulated
4-6-fold upon HOB cell adhesion to these matrix proteins (Fig. 1).
Generation of Human Osteoblastic Cell Lines Expressing a Dominant
Negative Erk1 Protein--
Since Erks mediate both growth factor- and
integrin- induced signal transduction, it is likely that they play
important role in the regulation of osteoblast function. We employed
the dominant negative Erk1 K71R (Erk1DN) protein, which can inhibit
both Erk1 and Erk2 activities (37), to analyze the role of Erks in
osteoblast function. Using a pseudotyped retrovirus as a carrier for
Erk1DN cDNA, 70-80% of transduced HOB cells expressed high level
of Erk1 mutant protein as compared with cells transduced with virus
encoding the LacZ protein (Fig. 2).
Western blot analysis using anti-pan-Erk antibody confirmed the
overexpression of Erk1 K71R in Erk1DN cells (Fig.
3, top panel,
top band at 44 kDa). Interestingly, the total amount of Erk2 was decreased to 68% of LacZ level in Erk1DN cells (Fig. 3, top panel, bottom
band at 42 kDa). Analysis of individual Erk activity by
Western blotting for p-Erk protein levels indicated that the
concentration of active p-Erk2 was further decreased to 30% of LacZ
level in Erk1DN cells (Fig. 3, second panel,
bottom band). Consistent with earlier reports
(35, 37), Erk1 K71R in Erk1DN cells could be phosphorylated by MEK
despite its defect in kinase activity (Fig. 3, second
panel, top band). Since p38 and JNK
are also members of the MAPK superfamily, we analyzed the activity of
these two kinases by Western blotting using antibodies that recognize
the activated forms of these enzymes. As shown in Fig. 3
(bottom three panels), active p38
(p-p38) and JNK (p-JNK) were not inhibited, but were, in fact,
stimulated (2-3-fold) by the expression of Erk1DN protein when
normalized with the total p38 levels. The stimulation of p38 activity
in Erk1DN cells is consistent with an earlier report that p38 activity
can be inhibited by Erk (41).
Analysis of total MAPK activity indicated that cells expressing Erk1DN
protein had lower (55%) basal MAPK activity as compared with LacZ
virus transduced cells (Figs. 4,
A and B, top left
two lanes). Since FGF-2, TGF- Expression of Erk1DN Protein Inhibited the Proliferation and
Differentiation of Human Osteoblastic Cells--
After establishing
the expression of functional Erk1DN protein, we examined the effect of
this protein on HOB cell proliferation. As shown in Fig.
6, the basal DNA synthesis, as measured
by [3H]thymidine incorporation, was slightly but
significantly decreased (86% of LacZ level) in Erk1DN cells. Since
both FGF-2 and TGF-
Since MAPK can regulate cell differentiation (26), we examined the
effects of Erk1DN protein on HOB cell differentiation by analyzing
differentiation markers including alkaline phosphatase activity, matrix
protein deposition, and matrix mineralization. Expression of Erk1DN
protein reduced alkaline phosphatase activity to 37% of the LacZ
control cell level (Fig. 7). In addition,
the protein levels of type I collagen, osteopontin, and bone
sialoprotein in the cell layers of Erk1DN cells were decreased to 47%,
63%, and 64%, respectively, of the corresponding LacZ level (Fig.
8). Consistently, matrix mineralization
was inhibited in Erk1DN cells after long term culture in the presence
of ascorbic acid and Expression of Erk1DN Protein Inhibited Cell Adhesion, Spreading,
Migration, and Integrin Expression--
Since Ras has been implicated
to regulate integrin activities, such as cell adhesion, spreading, and
migration, in an "inside-out" fashion (46-48) and since Erk is one
of the major downstream effectors of Ras, we examined the effect of
Erk1DN protein on these activities. Expression of Erk1DN protein
inhibited cell adhesion to type I collagen, osteopontin, and
vitronectin to 25%, 35%, and 31%, respectively, of the corresponding
LacZ level (Fig. 10). Cell spreading on
these matrices was also curtailed in Erk1DN cells (Fig.
11). Similarly, the migration of Erk1DN
cells on collagen, osteopontin, and vitronectin was reduced to 67%,
28%, and 26%, respectively, of the LacZ cell level (Fig.
12). Since integrins mediate cell
adhesion, spreading, and migration on these matrices, we examined the
surface integrin levels in Erk1DN and LacZ cells. As shown in Fig.
13, Erk1DN cells expressed lower levels
of We have demonstrated that the expression of an Erk1 dominant
negative protein inhibited Erk/MAPK activity, resulting in decreased proliferation and differentiation of human osteoblastic cells. The
expression of Erk1DN protein also inhibited osteoblast adhesion, spreading, migration, and integrin expression. These data indicate that
Erks of the MAPK superfamily play an important role in "outside-in" signal transduction regulating osteoblast growth and differentiation and in "inside-out" signal transduction modulating integrin levels on cell surface and, hence, cell adhesion, spreading, and migration. The inhibition of osteoblast function by Erk1DN protein was not a
result of global apoptosis since only a few dead cells (<0.01%) were
detected in Erk1DN cells by terminal dUTP nick-end labeling assay,
which was not significantly different from LacZ cells (data not shown).
The role of MAPK in cell proliferation and differentiation has been
well documented in a variety of cell systems (26). Our data demonstrate
that Erk/MAPK mediates FGF-2- and TGF- In addition to alkaline phosphatase, Erk/MAPK regulates the deposition
of bone matrix proteins (type I collagen, osteopontin, and bone
sialoprotein) and matrix mineralization by HOB cells. Type I collagen
and bone sialoprotein have been shown to serve as mineralization
scaffolds and hydroxyapatite initiation sites, respectively, whereas
osteopontin functions as the modulator of matrix mineralization
(51-54). Therefore, the inhibition of the deposition of these matrix
proteins by dominant negative Erk1 will retard matrix mineralization.
Recently, MEK, the up-stream effector of MAPK, has been shown to
regulate the activity of Cbfa1, a transcription factor that mediates
osteoblast differentiation (55). Stimulation of MAPK by constitutively
active MEK enhances the expression of osteocalcin, another osteoblast
differentiation marker, whereas the dominant negative MEK or MEK
inhibitor PD98059 inhibits osteocalcin expression (55). These
observations suggest that MAPK is essential for osteoblast
differentiation and matrix mineralization.
Bone is a dynamic tissue that undergoes constant remodeling. For bone
formation to occur, osteoblast precursor cells must migrate from
residing bone marrow compartment to the remodeling site where they
differentiate. The inhibition of integrin expression by Erk1DN suggests
that the Erk/MAPK pathway is critical for osteoblast integrin
expression, which, in turn, dictates cell migration, adhesion, and
spreading on matrices since integrins play important role in these
processes. MAPK has previously been shown to be associated with the
up-regulation of integrins (56, 57). Our data provide direct evidence
linking Erk/MAPK signaling to the expression of Several mechanisms may mediate the inhibition of osteoblast activity by
Erk1DN protein. Among these, the inhibition of AP-1 activity appears to
be a critical factor (58). It is important to note that the expression
of type I collagen, osteocalcin, and osteopontin has been shown to be
dependent on AP-1 (59-61). Other transcription factors may also
contribute to the Erk/MAPK effects, especially the aforementioned Cbfa1
since the activation of Cbfa1 is dependent on Erk/MAPK (26, 44, 55).
Cbfa1 can regulate the expression of bone sialoprotein, type I
collagen, osteocalcin, and osteopontin in osteoblasts (62-65).
Therefore, the inhibition of Erk activity in Erk1DN cells will prevent
the activation of Cbfa1 leading to the inhibition of bone matrix
protein deposition.
Recently, the mutated gene for Coffin-Lowry syndrome (CLS) has been
identified as ribosomal S6 kinase 2 (Rsk2) (66, 67). This syndrome is
characterized by a variety of clinical symptoms including mental
retardation, delayed closure of fontanelles, digital dysmorphism,
progressive skeletal deformations, osteopenia, and delayed bone age
(68-71). Since Rsk2 is a substrate that is phosphorylated and
activated by Erk (44), the skeletal defects observed in CLS patients
suggest that Rsk2 is one of the important down-stream effectors in the
regulation of osteoblast function by Erk. The inhibition of RSK2
activity in Erk1DN cells is consistent with this hypothesis. Our data
also provide an explanation for the skeletal abnormalities observed in
patients with CLS since lack of RSK2 may result in inhibition of
osteoblast growth, differentiation, adhesion, spreading, and migration.
1,
v
3, and
v
5 integrins on the surface was decreased. Metabolic labeling indicated that the synthesis of these
integrins was inhibited in Erk1DN cells. These data suggest that Erks
are not only essential for the growth and differentiation of
osteoblasts but also are important for osteoblast adhesion, spreading,
migration, and integrin expression.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, and BMP-2
not only are potent chemotactic agents for osteoblasts but also have
profound effects on their growth and differentiation (1-5). In
addition to growth factors, the interaction between integrins and the
extracellular matrix proteins also governs cell migration, adhesion,
proliferation, and differentiation (6-10). Human osteoblastic (HOB)
cells express a repertoire of integrins, which interact with bone
matrix proteins such as osteopontin, bone sialoprotein, vitronectin,
type I collagen, and fibronectin (11-15). These interactions have been
shown to regulate osteoblast growth and differentiation (16-23).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP, ECL kit, and protein G-Sepharose were from
Amersham Pharmacia Biotech. AP-1 consensus oligonucleotide
(5'-CGCTTGATGAGTCAGCCGGAA-3') and T4 polynucleotide kinase were from
Promega (Madison, WI). Recombinant protein A-Sepharose 4B was from
Zymed Laboratories Inc. (San Francisco, CA).
Polyclonal antibodies against type I collagen
chain (LF-67),
osteopontin (LF-7), and bone sialoprotein (LF-6) were kindly provided
by Dr. Larry W. Fisher (National Institutes of Health, Bethesda, MD).
Vitronectin and monoclonal antibody against human
v
5 (P1F6) were from Life Technologies,
Inc. Monoclonal antibodies against human
v
3 (LM609) and human
1
were purchased from Chemicon International Inc. (Temecula, CA). The
anti-pan-Erk antibody, which recognizes the C-terminal half of Erk1 and
Erk2 molecules, was from Transduction Laboratories (Lexington, KY). Polyclonal antibody against phosphorylated Erk (p-Erk) was from New
England Biolabs, Inc. (Beverly, MA). Monoclonal antibodies against p38,
phosphorylated p38 (p-p38), and phosphorylated JNK (p-JNK), and goat
anti-RSK2 antibody were from Santa Cruz Biotechnology, Inc. (Santa
Cruz, CA). MAPK assay kit was from Stratagene (La Jolla, CA). S6 kinase
assay kit was from Upstate Biotechnology (Lake Placid, NY). Transwells
with polycarbonate membrane containing 8.0-µm pores were from Costar
(Cambridge, MA). Phosphatase inhibitor mixtures I (p2850) and II
(p5726), protease inhibitor mixture (p8340), and anti-tubulin antibody
were from Sigma. Leukostat kit was from Fisher.
-minimum Eagle's medium (
-MEM) supplemented with
10% heat-inactivated fetal bovine serum (HIFBS). Since the results
obtained with either osteoblasts or bone marrow stromal cells were
similar, we defined these two cell types together as human osteoblastic
(HOB) cells for convenience.
5 × 106 colony-forming units/ml) were combined and
used for transduction. Before transduction, the medium was filtered
through a 0.45-µm membrane and Polybrene (hexadimethrine bromide, 8 µg/ml) was added to the virus medium. As a negative control, a virus
carrying the SFG-LacZ cDNA was generated in the same fashion. This
pseudotyped retrovirus has been shown to have no effect on osteoblast
differentiation (40).
-MEM with 10% HIFBS.
-MEM containing 0.2% HIFBS for 24 h,
followed by treatment with designated growth factor for 10 min at
37 °C. After two PBS washes, the cell layers were extracted with 0.5 ml of MAPK assay buffer consisting of 50 mM Tris-HCl, pH
7.4, 150 mM NaCl, 5 mM EDTA, 1% Nonidet P-40,
5 mM NaF, phosphatase inhibitor mixture II (0.4 ml/10 ml),
and protease inhibitor mixture (0.4 ml/10 ml). Extracts were
homogenized by passing through 26-gauge needles 10 times. After
microcentrifugation, the supernatant was transferred to a new tube.
MAPK activity in the cell extract (20 µg of protein) was measured by
using the MAPK assay kit with PHAS-1 protein as the substrate and
[
-32P]ATP according to the protocol supplied by the
manufacturer (Stratagene).
-32P]ATP, was incubated with nuclear extracts (2 µg) for 20 min in binding buffer which consisted of 50 mM
Tris-HCl, pH 7.5, 250 mM NaCl, 2.5 mM EDTA, 2.5 mM DTT, 20% glycerol, and 0.25 mg/ml poly(dI-dC). The
binding reaction was terminated by the addition of 1 µl of 5× TBE
buffer (0.445 M Tris borate, pH 8.3, and 10 mM
EDTA) containing 0.03% bromphenol blue, 0.03% xylene cyanol, and 30%
glycerol. The mixtures were separated on 4-20% polyacrylamide gradient gels in 0.3× TBE. The gels were dried and subjected to autoradiography.
-glycerophosphate, 25 µg/ml aprotinin, 10 µg/ml each leupeptin and pepstatin, 50 µg/ml benzamidine, 1 mM PMSF, and phosphatase inhibitor mixture I (1:100).
Supernatants (0.6 mg) obtained after microcentrifugation were incubated
with goat anti-RSK2 antibody (1:200) together with protein G-Sepharose
at 4 °C overnight. The beads were collected by microcentrifugation,
washed twice with lysis buffer, followed by kinase buffer (20 mM MOPS, pH 7.2, 25 mM
-glycerophosphate, 5 mM EGTA, 1 mM Na3VO4,
and 1 mM DTT). RSK2 phosphotransferase activity in the
beads was measured by using S6 kinase assay kit and
[
-32P]ATP according to the protocols provided by the
manufacturer (Upstate Biotechnology, Inc.).
-MEM with 10% HIFBS. The medium was removed, and cells
were incubated in 0.2% HIFBS medium for another 24 h.
[3H]Thymidine (2 µCi/well) in
-MEM containing 0.2%
HIFBS was added with the appropriate growth factor and the incubation
continued for another 24 h. [3H]Thymidine
incorporated into DNA was measured after trichloroacetic acid
precipitation, ethanol washing, and alkali extraction as described
previously (34).
-MEM, 10% HIFBS, ascorbic acid (50 µg/ml)
and
-glycerophosphate (10 mM). At the end of incubation,
cells were washed three times with PBS and fixed in ice-cold 70%
ethanol for 1 h. After washing with water three times, the cells
were incubated with 0.4% alizarin red-S in water for 10 min at room
temperature, followed by five washes with water and final incubation in
PBS for 15 min.
-MEM supplemented with 0.1% BSA, cells were seeded to wells
pre-coated with either BSA or a matrix protein (4 × 104 cells/cm2). After 1 h of incubation
for adhesion measurement or 2 h for spreading assay at 37 °C,
the wells were washed with PBS to remove the non-adherent cells.
Adherent cells were fixed and stained with 0.5% toluidine blue in 4%
paraformaldehyde. To obtain the adherent cell number, stained cells
were extracted with 1% SDS, and the absorbance at 630 nm was measured.
For analysis of cell spreading, cell morphology was recorded by
photography using phase contrast microscope. For adhesion-induced MAPK
assays, cells were adhered to matrices for 20 min and lysed in MAPK
assay buffer as described above.
-MEM with 0.1% BSA and 25 ng/ml FGF-2. After overnight migration in
an incubator, cells on the membranes were fixed and stained using a
Leukostat kit. The non-migrated cells on top of the membranes were
removed with cold PBS-soaked cotton swabs, and the migrated cells on
the bottom side of the membranes were counted under the microscope with
the aid of a grid. A total of nine separate fields (3 × 10
4 cm2/field) were counted for
each sample, and the average number of cells per field was calculated.
1,
v
3,
or
v
5 and protein A-Sepharose at 4 °C
on a Nutator. Pellets obtained after microcentrifugation were washed
twice with radioimmune precipitation assay buffer, followed by PBS
containing 0.5% Tween 20 and 0.02% NaN3 in addition to 1 mg/ml ovalbumin, and finally with PBS containing 0.5% Tween 20 and
0.02% NaN3. Immunoprecipitated integrins in the beads were
extracted with sample buffer and applied to SDS-PAGE. Integrin bands
were visualized by autoradiography. For measurement of integrin
biosynthesis, cells were incubated in methionine- and cystine-
deficient medium containing 5% dialyzed HIFBS and 25 µCi/ml
Tran35S-Label for 24 h. After lysis,
35S-labeled integrins were analyzed by immunoprecipitation
and SDS-PAGE as described above.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Activation of Erk/MAPK activity by adhesion
of HOB cells to bone matrix proteins. HOB cells were adhered to
the indicated bone matrix protein or remained in suspension for 20 min.
Cells were extracted, and aliquots containing equal amount of protein
were subjected to Western blot analysis for measurement of activated
Erk (p-Erk) and tubulin levels. MAPK activity was also assayed by using
PHAS-1 as the substrate and [ -32P]ATP followed by
SDS-PAGE and autoradiography.
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Fig. 2.
Expression of Erk1 K71R in HOB cells.
HOB cells were transduced with a pseudotyped retrovirus encoding either
LacZ protein (top) or Erk1 K71R protein (bottom).
After 48 h, cells were fixed and stained with anti-pan-Erk
antibody, which recognizes both native Erk and Erk1K71R proteins.
Similar results were obtained when anti-Erk1 antibody was
employed.
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Fig. 3.
Effect of Erk1DN on Erk, JNK, and p38
activity in HOB cells. Cells were transduced with virus encoding
either LacZ or Erk1 K71R (Erk1DN) protein. After confluence, cells were
extracted and Western blot analysis performed using antibodies against
pan-Erk, p-Erk, p-p38, p38, or p-JNK.
, and BMP-2 can
stimulate MAPK activity in osteoblastic cells (27, 42,
43),2 we also analyzed the
MAPK activity in transduced cells after stimulation with these growth
factors. As shown in Fig. 4A, the FGF-2-stimulated MAPK
activity in Erk1DN cells was only 22% of LacZ cell level under the
same conditions (Fig. 4A, top right two lanes). Similarly, the stimulation of MAPK
activity by BMP-2 and TGF-
in Erk1DN cells was only 30% of the
values obtained from stimulated LacZ cells (Fig. 4B,
top right four lanes). Fig. 4 (bottom panels of A and
B) showed the total Erk proteins in the extracts, further
confirming the expression of Erk1DN protein in cells transduced with
Erk1DN viruses. Since activation of Erks leads to increased c-Fos
transcription and AP-1 activity (26), we examined the effect of Erk1DN
protein on AP-1 activity in osteoblasts. Results of EMSA demonstrated
that the expression of Erk1DN protein strongly inhibited AP-1 binding
activity to its consensus oligonucleotide to 10% of LacZ control cell
level (Fig. 4C). Moreover, the activity of RSK2, another
downstream effector of Erk (44), was inhibited by 54% in Erk1DN cells
as compared with the LacZ cells after normalization for protein
loading, both under basal condition and after FGF-2 stimulation (Fig.
5). These combined data indicated that
Erk1 K71R cDNA introduced into HOB cells by using pseudotyped
retrovirus was expressed and was functional in both basal and growth
factor-stimulated Erk1DN cells.
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Fig. 4.
Inhibition of growth factor-activated MAPK
activity and AP-1 activity by Erk1DN. HOB cells were transduced
with virus encoding either LacZ (L) or Erk1DN (E)
protein. After confluence, cells were stimulated with vehicle, FGF-2
(50 ng/ml), BMP-2 (100 ng/ml), or TGF- (1 ng/ml) for 10 min. Cell
layers were extracted and MAPK activity measured by the phosphorylation
of PHAS-1. Total Erk concentration in the extracts was measured by
Western blot analysis using anti-pan-Erk antibody. A, MAPK
activity before and after stimulation with FGF-2. B, MAPK
activity before and after stimulation with BMP-2 or TGF-
.
C, nuclear extracts derived from LacZ (L) or
Erk1DN (E) cells were incubated with radiolabeled AP-1
consensus oligonucleotide and EMSA performed.
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Fig. 5.
Inhibition of RSK2 activity by Erk1DN.
Cells transduced with virus encoding either LacZ or Erk1DN protein were
stimulated with either vehicle (Control) or FGF-2 (50 ng/ml)
for 15 min. RSK2 activity in the cell lysate was measured by using the
S6 kinase assay kit and [ -32P]ATP after
immunoprecipitation with anti-RSK2 antibody. Inset, Western
blotting of total RSK2 for loading variation in kinase assay.
L, LacZ cells; E, Erk1DN cells. *,
p < 0.01 when compared with the corresponding LacZ
level.
stimulate the proliferation of normal human
osteoblastic cells (4, 45), we analyzed the effects of Erk1DN protein
on the proliferation induced by these factors. As expected, FGF-2
stimulated the incorporation of [3H]thymidine into DNA in
LacZ cells (135% of the basal level), whereas no stimulation of DNA
synthesis was detected in Erk1DN cells (Fig. 6). Similarly, TGF-
augmented DNA synthesis in LacZ cells (186% of the basal level)
whereas this stimulation was diminished in Erk1DN cells (129% of the
basal level) (Fig. 6).
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Fig. 6.
Erk1DN inhibits osteoblast
proliferation. HOB cells transduced with virus encoding either
LacZ or Erk1DN protein were stimulated with vehicle (Basal),
FGF-2 (50 ng/ml), or TGF- (1 ng/ml). Cell proliferation was measured
by [3H]thymidine incorporation into DNA. a,
p < 0.05 when compared with the corresponding LacZ
cell level. b, p < 0.05 when compared with
the corresponding basal level.
-glycerophosphate (Fig.
9). Thus, expression of Erk1DN protein
inhibits both proliferation and differentiation in HOB cells.
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Fig. 7.
Erk1DN inhibits alkaline phosphatase
activity. HOB cells transduced with virus encoding either Erk1DN
or LacZ protein were allowed to grow to confluence. Alkaline
phosphatase activity in the cell layer was measured and normalized with
total cellular protein concentration.
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Fig. 8.
The bone matrix protein levels are reduced in
Erk1DN cells. HOB cells transduced with virus encoding either LacZ
or Erk1DN protein were allowed to grow to confluence. The levels of
type I collagen, osteopontin, and bone sialoprotein in the cell layer
were measured by Western blot analysis and quantified by densitometry
analysis of the bands on x-ray films.
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Fig. 9.
Inhibition of matrix mineralization by
Erk1DN. HOB cells transduced with virus encoding either LacZ or
Erk1DN protein were cultured in the presence of -glycerophosphate
and ascorbic acid for 28 days. Mineralized matrix was visualized by
alizarin red-S staining.
1 (43%),
v
3 (54%),
and
v
5 (51%) integrins when compared
with the LacZ cell levels. Metabolic labeling with
Tran35S-Label indicated that the synthesis of
1,
v
3, and
v
5 integrins in Erk1DN cells was only
77%, 70%, and 64%, respectively, of the corresponding LacZ cell
level (Fig. 14). These results
suggested that the inhibition of integrin expression on the cell
surface was derived, at least in part, by the inhibition of integrin
synthesis.
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Fig. 10.
Erk1DN inhibits osteoblast adhesion to bone
matrix proteins. HOB cells transduced with virus encoding either
LacZ or Erk1DN protein were adhered to type I collagen, osteopontin, or
vitronectin for 1 h. The number of adherent cells was measured by
absorbance at 630 nm after fixation, staining with toluidine blue, and
solubilization of the blue dye with SDS solution. a,
p < 0.001 when compared with the corresponding LacZ
level.
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Fig. 11.
Erk1DN inhibits osteoblast spreading on bone
matrix proteins. HOB cells transduced with virus encoding either
LacZ or Erk1DN protein were allowed to adhere and spread on type I
collagen, osteopontin, or vitronectin for 2 h. Cells were fixed
and visualized under microscope.
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Fig. 12.
Erk1DN inhibits osteoblast migration on bone
matrix proteins. HOB cells transduced with virus encoding either
LacZ or Erk1DN protein were allowed to migrate on type I collagen,
osteopontin, or vitronectin in the presence of FGF-2 (25 ng/ml) for
24 h in Costar transwells. Migrated cells at the bottom of the
membranes were counted. a, p < 0.001 when
compared with the corresponding LacZ level.
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Fig. 13.
Reduction of surface integrin levels by
Erk1DN. Cells transduced with virus encoding either LacZ
(L) or Erk1DN (E) protein were allowed to grow to
confluence. Levels of 1,
v
3, and
v
5
integrins on cell surface were measured after labeling with
125I followed by immunoprecipitation with specific antibody
and protein A-Sepharose pull-down. Immunoprecipitated integrins were
separated by SDS-PAGE, visualized by autoradiography, and quantified by
scanning the autoradiograms.
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Fig. 14.
Erk1DN inhibits integrin synthesis.
Cells transduced with virus encoding either LacZ (L) or
Erk1DN (E) protein were allowed to grow to confluence. The
day before harvest, cells were metabolically labeled with
Tran35S-Label. Newly synthesized 1,
v
3, and
v
5
integrins were measured after immunoprecipitation, SDS-PAGE, and
autoradiography.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-stimulated proliferation in
human osteoblastic cells. In addition, our observation that Erk/MAPK is
activated by osteoblast adhesion to several bone matrix proteins, such
as type I collagen, fibronectin, vitronectin, and osteopontin, is
consistent with earlier reports using other cell systems (6, 8-10). In
osteoblast differentiation, alkaline phosphatase activity is increased
early in the process, and it is this increase that commits the cells to
differentiate into osteoblast lineage (49). A shortage or complete
deficiency in alkaline phosphatase activity delays or prevents matrix
mineralization in osteoblasts (50). In line with these reports,
inhibition of Erk activity in osteoblasts decreases not only alkaline
phosphatase activity but also matrix mineralization. Thus, Erk appears
to be essential for the commitment of osteoblasts to differentiate, and
our results are consistent with the role of alkaline phosphatase in
matrix mineralization. It has been reported that the stimulation of
MAPK activity upon adhesion of osteoblasts to collagen is via FAK
signaling and that it is this stimulation of FAK that leads to elevated
alkaline phosphatase mRNA level (32). Since Erk/MAPK is stimulated
by adhesion of HOB cells to various bone matrix proteins, and since
Erk/MAPK is one of the down-stream effectors of FAK, the decreased
alkaline phosphatase activity observed in Erk1DN cells may stem from
the modulation of FAK/MAPK signaling.
1,
v
3, and
v
5
integrins. Although the reduction of surface integrin levels in Erk1DN
cells can be attributed in part to the decreased synthesis, the latter
is only to a moderate degree (23-36% inhibition) as compared with the
former (46-57% reduction). Therefore, other mechanism(s) may also be
regulated by Erk/MAPK to affect the integrin levels on cell surface.
The extent of inhibition on cell adhesion and migration in Erk1DN cells
(~70% inhibition with the exception of migration on collagen) cannot
be fully accounted for by the degree of reduction in surface integrin
levels. These data suggest that Erk may affect the integrin activity.
Additional studies will be required to confirm these speculations.
Since cell migration is preceded by cell adhesion, the inhibition of
cell migration in Erk1DN cells may simply be the consequence of
decreased cell adhesion. In addition, cells need to spread
directionally toward chemokine during migration, the decreased ability
of Erk1DN cells to spread will hinder cell migration.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Melanie H. Cobb for providing us the Erk1 K71R plasmid, Dr. Larry W. Fisher for antibodies against bone matrix proteins, and the Genetics Institute (Cambridge, MA) for BMP-2.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants AR32087 and AR07033.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.
This paper is dedicated to the memory of Dr. Louis V. Avioli.
To whom all correspondence should be addressed: Div. of Bone
and Mineral Diseases, Barnes-Jewish Hospital of St. Louis, Washington University School of Medicine, 216 S. Kingshighway Blvd., St. Louis, MO
63110. Tel.: 314-454-8406; Fax: 314-454-5047; E-mail: scheng@im.wustl.edu.
Published, JBC Papers in Press, January 29, 2001, DOI 10.1074/jbc.M010021200
2 C.-F. Lai and S.-L. Cheng, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
FGF, fibroblast
growth factor;
TGF, transforming growth factor;
Erk, extracellular
signal-regulated kinase;
Erk1DN, dominant negative Erk1;
HIFBS, heat-inactivated fetal bovine serum;
HOB cell, human osteoblastic cell;
MAPK, mitogen-activated protein kinase;
-MEM,
-minimum Eagle's
medium;
PBS, phosphate-buffered saline;
PT, phosphate-buffered saline
with 0.1% Triton X-100;
DTT, dithiothreitol;
PMSF, phenylmethylsulfonyl fluoride;
PAGE, polyacrylamide gel
electrophoresis;
CLS, Coffin-Lowry syndrome;
MOPS, 4-morpholinepropanesulfonic acid;
BMP, bone morphogenetic protein;
BSA, bovine serum albumin;
JNK, c-Jun N-terminal kinase;
FAK, focal adhesion
kinase;
EMSA, electrophoretic mobility shift assay.
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REFERENCES |
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