The Protein Kinase C Pathway Plays a Central Role in the
Fibroblast Growth Factor-stimulated Expression and Transactivation
Activity of Runx2*
Hyun-Jung
Kim
,
Jung-Hwan
Kim
,
Suk-Chul
Bae§,
Je-Yong
Choi¶,
Hyun-Jung
Kim
, and
Hyun-Mo
Ryoo
**
From the
Department of Biochemistry, School of
Dentistry and Biomolecular Engineering Center, the ¶ Department of
Biochemistry, School of Medicine, and the
Department of
Pediatric Dentistry, Kyungpook National University, University,
Daegu, 700-422, Korea and the § Department of Biochemistry,
School of Medicine, Chungbuk National University, Chungju
361-373, Korea
Received for publication, April 18, 2002, and in revised form, October 1, 2002
 |
ABSTRACT |
Fibroblast growth factor (FGF)/FGF receptor
(FGFR) signaling induces the expression of Runx2, a key transcription
factor in osteoblast differentiation, but little is known about the
molecular signaling mechanisms that mediate this. Here we examined the
role of the protein kinase C (PKC) pathway in regulating Runx2 gene expression and its transactivation function. Treatment with FGF2 or
FGF4, or transfection with a vector expressing a mutant FGFR2 that is
constitutively activated in the absence of ligand, strongly stimulates
Runx2 expression. Electrophoretic mobility shift assays also showed
that FGF2 treatment increases the specific binding of Runx2 to the
cognate response element in the osteocalcin gene promoter. Blocking PKC
completely inhibited FGF2-induced Runx2 expression, whereas
mitogen-activate protein kinase inhibitors had no effect. The
FGF/FGFR-stimulated 6xOSE2 promoter activity was also blocked by
inhibiting PKC, as was the FGF2 stimulation of the DNA-binding activity
of Runx2. Experiments with PKC isoform-specific inhibitors and
dominant negative isoforms of PKC indicate that PKC
is
one of key isoforms involved in the FGF2-stimulated Runx2 expression.
In addition, experiments with Runx2-knockout cells showed
that, although the PKC pathway largely regulates FGF2-stimulated Runx2
activity by up-regulating Runx2 expression, it also modifies Runx2
protein post-translationally and thereby increases its transcriptional activity. Thus, we show for the first time that FGF/FGFR signaling stimulates the DNA-binding and transcriptional activities of Runx2 as
well as its expression, and these are largely regulated by the PKC pathway.
 |
INTRODUCTION |
Fibroblast growth factors
(FGFs)1 are important
autocrine/paracrine signaling molecules involved in intramembranous and
endochondral bone formation. They include FGF2, which is a strong
mitogen for bone-derived cells (1-5). Disruption of the
FGF2 gene results in decreased bone formation (6), whereas
overexpression of FGF2 in transgenic mice causes achondroplasia, the
premature transformation of cartilage to bone (7). Constitutive active
mutations of FGF receptors (FGFR) 1, 2, and 3 caused a
congenital abnormality, craniosynostosis, which is characterized by
premature osseous obliteration of cranial suture (8, 9). In
osteoblasts, the interaction of FGFs like FGF2 with their FGFRs induces
receptor dimerization and autophosphorylation of receptors, which in
turn activates multiple signal transduction pathways, including those involving mitogen-activated protein kinases (MAPKs) and protein kinase
C (PKC) (10-13). However, the signaling pathways that mediate the
biological actions of FGF/FGFR in osteoblasts remain unclear.
Runt-related transcription factor 2 (Runx2), previously
known as
Cbfa1/Pebp2
A/AML3, plays
an essential role in osteoblast differentiation (14-16).
Runx2-knockout animals display complete bone loss because of
arrested osteoblast maturation (15) while heterozygotes develop
cleidocranial dysplasia, which is characterized by delayed ossification
(16, 17). Several reports show that Runx2 regulates the expression of
bone marker genes during osteoblast differentiation (14, 18, 19). In
our previous report, we investigated the expression pattern of Runx2
during intramembranous bone formation and found that Runx2 expression
is needed for all steps of osteoblast differentiation, namely, from the
commitment to differentiate through to the final differentiation event
(20). Taken together, these observations clearly indicate that Runx2 plays a key role in osteoblast differentiation.
In humans, a mutation in FGFR1 that causes its constitutive
activation is associated with Pfeiffer syndrome, one of the syndromes that comprise craniosynostosis as a phenotype. In mice, gene
replacement with a constitutively active FGFR1 mutant
results in increased expression of Runx2 and premature suture closure
that is the representative phenotype of human craniosynostosis (21).
This suggests that Runx2 may be a downstream target gene in FGF/FGFR
signaling during bone formation. However, the mechanisms by which
activated FGFRs increase Runx2 expression are not yet understood. In
this report, we demonstrate for the first time that FGF/FGFR
stimulation of Runx2 expression, which leads to the transactivation of
downstream genes, is mediated by the PKC pathway.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Recombinant human FGF2 and the luciferase assay
system were purchased from Promega (Madison, WI). Recombinant human
FGF4 was obtained from R&D Systems (Minneapolis, MN). Calphostin C and bovine serum albumin (BSA) were purchased from Sigma Chemical Co. (St.
Louis, MO), and Zeta-probe blotting membranes and the Bradford protein
assay kit were from Bio-Rad (Hercules, CA).
-MEM, DMEM, and
LipofectAMINE Plus reagent were purchased from Invitrogen (Grand
Island, NY). Fetal bovine serum (FBS) and ExpressHyb hybridization solution were obtained from HyClone (Logan, UT) and
Clontech (Palo Alto, CA), respectively. GF109203X
was from BIOMOL (Plymouth Meeting, PA). Rottlerin and Go6976 were from
Calbiochem (San Diego, CA). The p38 MAPK inhibitor SB203580 and
the MEK1 inhibitor PD98059 were purchased from Tocris (Ballwin, MO) and
were prepared as 25 and 50 mM stock solutions in
Me2SO, respectively.
Cell Culture--
Osteoblast-like MC3T3-E1 cells (22) were
maintained in
-MEM supplemented with 10% FBS, 100 units/ml
penicillin, and 100 µg/ml streptomycin at 37 °C in a humidified
atmosphere with 5% CO2. Murine premyoblast C2C12 cells
(23) were maintained in DMEM containing 15% FBS, whereas rat
osteosarcoma ROS17/2.8 cells (22) were cultured in DMEM with 10% FBS.
Runx2
/
calvaria cell lines were maintained in
-MEM
supplemented with 10% FBS (24). Cells were washed twice with
phosphate-buffered saline (PBS) and then treated for 3 h with 10 ng/ml FGF2 or FGF4 in serum-free medium supplemented with 0.2% BSA. In
some experiments, the cells were pretreated for 1 h with
calphostin C (0.5 µM), GF109203X (1 µM),
Go6976 (200 nM) or rottlerin (1-12 µM),
inhibitors of protein kinase C, or 50 µM PD98059, a
MEK1/2 specific blocker, or 25 µM SB203580, a p38
specific inhibitor. Thereafter, they were treated with 10 ng/ml FGF2.
RNA Preparation and Northern Blot Analysis--
Total RNA was
prepared by single-step isolation as previously described (22), and the
concentration was determined by spectrophotometry. Total RNA (10 µg)
was then heated to 65 °C for 15 min in 50% formamide, 0.02%
formaldehyde, 40 mM MOPS, and 0.1 mg/ml ethidium bromide prior to gel electrophoresis on a 1% agarose gel containing 5.5% formaldehyde and 40 mM MOPS. The RNA was then blotted onto
a Zeta-probe membrane in 20× SSC. The blot was air-dried and
cross-linked by exposure to ultraviolet light. Runx2 cDNA was
labeled with [
-32P]dCTP by using the Megaprime DNA
labeling system kit (Amersham Biosciences, UK). Prehybridization and
hybridization were performed by using the ExpressHyb solution according
to the manufacturer's protocol. After hybridization, the membrane was
washed in 2× SSC/0.1% SDS at room temperature and then in 0.1×
SSC/0.1% SDS at 50 °C and exposed to x-ray film at
70 °C with
intensifying screens.
Transient Transfection and Luciferase Assay--
Cells were
transfected by following the protocol recommended by the manufacturers
of the LipofectAMINE Plus reagent. Cells were cultured in six-well
plates at a density of 1 × 105 cells/well for 16-24
h and then transfected with the reporter vector (1 µg/well) and/or
the expression vector (1 µg/well) in serum-free medium. Three hours
later, the medium was replaced by medium containing 10% FBS and
cultured overnight. The medium was then replaced with serum-free medium
with or without FGF2, and the incubation was continued for 16-24 h.
Cell lysates were prepared and luciferase activity was determined with
the Luciferase Assay System kit (Promega). The luciferase activity was
normalized with respect to the protein content as determined by the
Bradford protein assay kit (Bio-Rad). Luminescence was measured by an
AutoLumat LB953 instrument (EG&G Berthold, Walloc, Finland). For the
forced expression of Runx2, we used the Runx2-type II expression
vector, which translates a 57-kDa product (25). Expression
vector for constitutive active FGFR2 (FR2Y340H) causing human Crouzon
syndrome and dominant negative PKC
were provided by Dr. Donoghue
(26) and Dr. Soh (27), respectively. The promoter region of rat
osteocalcin (
208/+23) was subcloned into pGL2-basic vector from
208
OC-CAT and
208 Runx mut vector (25).
Preparation of Nuclear Extract and Electrophoretic Mobility Shift
Assays--
Nuclear extracts were prepared as previously described
(28). For electrophoretic mobility shift assays, previously
characterized wild type and mutant OSE2 oligonucleotides corresponding
to the
156/
112 segment of the mouse osteocalcin promoter (29) were used. Probes were labeled with [
-32P]dCTP.
Approximately 1 ng of labeled oligonucleotide probe was incubated with
10 µg of nuclear extract for 30 min at room temperature in the
presence of 1× gel shift binding buffer containing 15 mM Tris, pH 7.5, 1 mM MgCl2, 0.5 mM
EDTA, 0.5 mM dithiothreitol, 50 mM NaCl, 10%
glycerol, and 1 µg of poly(dI-dC). Competition was performed with a
10- to 50-fold molar excess of unlabeled OSE2. Antibodies against Runx2
were preincubated with nuclear extracts and binding buffer for 30 min
at 4 °C prior to the addition of the probe. Reactions were separated
on 5% non-denaturating polyacrylamide gel.
Construction of the p6xOSE2-Luc Reporter Plasmid and
Establishment of a Stable p6xOSE2-Luc-transfected Cell Line--
To
construct the p6xOSE2-Luc reporter plasmid containing six tandem
repeats of the osteoblast-specific core binding sequences (OSE2),
oligonucleotides including PuACCPuCA were synthesized for sense and for
antisense in such a way as to create XmaI-cohesive overhangs
at each 5'-end as shown as follows: sense,
5'-CCGGGCTGCAATCACCAACCACAGCATC-3'; antisense,
3'-CGACGTTAGTGGTTGGTGTCGTAGGGCC-5'.
Annealed fragments containing OSE2 were multimerized by T4 DNA ligase.
6xOSE2 multimers were separated and purified by agarose gel
electrophoresis and then cloned into the XmaI site of the pGL2-promoter reporter vector (Promega). To confirm that the 6xOSE2 multimers were tandemly repeated, the cloned fragment was
sequenced. To establish a stable p6xOSE-Luc-transfected cell line,
C2C12 myogenic stem cells were co-transfected with the p6xOSE2-Luc
reporter vector and pcDNA3 in a 5:1 ratio with the LipofectAMINE
reagent according to the manufacturer's instructions. Because
FGF2-mediated stimulation of Runx2 expression, DNA binding, and
transactivation function of Runx2 protein in C2C12 cells was not
different from that in MC3T3-E1 cells (Fig. 1A), and these
cells can be differentiated into osteoblasts in response to osteogenic
signals (23, 24), C2C12 cells were selected as the host of this stable
transfection. Stably transfected cells were selected in media
containing 2.5 mg/ml G418 (Promega). The 6xOSE2 promoter activity of
each clone was assayed by using the luciferase assay system. Once
established, the clones were maintained in media containing 100 µg/ml G418.
Western Blot Analysis--
Nuclear extracts were resolved by
SDS-PAGE and electrotransferred to a polyvinylidene difluoride
membrane. After transfer, the blot was washed with PBS for 5 min at
room temperature and then incubated in blocking buffer (1× PBS
containing 0.1% Tween 20 with 5% nonfat dry milk) for 1 h at
room temperature. The blot was washed three times for 5 min each with
1× PBS. Primary antibody (Runx2) (24) was added to the blocking buffer
at 1:2000 dilution with gentle agitation overnight. The blot was washed
three times with PBS and incubated with the horseradish
peroxidase-conjugated rabbit anti-mouse antibody (Advanced
Biochemicals, Inc., Seoul, Korea) for 1 h at room
temperature. After three washes, the signal was detected by ECL plus
(Amersham Biosciences, UK).
 |
RESULTS |
FGF/FGFR Signaling Increases Runx2 mRNA
Levels--
Murine osteoblast-like MC3T3-E1 cells, rat osteosarcoma
ROS17/2.8 cells, and murine premyoblastic C2C12 cells were treated with
recombinant human FGF2. Although the basal expression levels of Runx2
were different in each cell type, FGF2 treatment stimulated Runx2
expression in all three cell lines (Fig.
1A). It is noteworthy that
each cell line expressed different Runx2 isoforms, because MC3T3-E1
cells mainly expressed the Runx2-type II isoform, ROS 17/2.8 cells
mainly expressed the Runx2-type I isoform, and C2C12 cells expressed
both isoforms equally. This isoform composition was not altered by FGF2
treatment. FGF4, another FGF family member, also increased Runx2
mRNA expression to a similar extent as FGF2 in C2C12 cells (Fig.
1B) and MC3T3-E1 cells (data not shown). MC3T3-E1 cells
transfected with an expression vector expressing the constitutively
active mutant of FGFR2 (FR2Y340H) also stimulated Runx2 mRNA
expression, even in the absence of FGF treatment (Fig. 1C).
Thus, Runx2 expression is up-regulated by FGF/FGFR signaling.

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Fig. 1.
FGF/FGFR signaling up-regulates Runx2
expression. A, MC3T3-E1 osteoblast-like cells
(MC), ROS17/2.8 rat osteosarcoma cells (ROS), and
C2C12 mouse premyoblastic cells were cultured until they reached early
confluence. The cells were cultured for an additional 3 h with or
without FGF2 in BSA-supplemented serum-free medium. Runx2 expression
was determined by Northern blot analysis. B, C2C12 cells
were treated with FGF2 or FGF4 in BSA-supplemented serum-free medium.
Runx2 expression was determined by Northern blot analysis.
C, MC3T3-El cells were transfected with a vector expressing
a constitutively active mutant of FGFR2 (FR2Y340H, derived from the
human Crouzon syndrome) or with empty vector (mock) as a
control. Runx2 expression was determined by Northern blot analysis.
Ribosomal RNA (rRNA) is shown as control for equivalent
loading of RNA.
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|
FGF2 Enhances the DNA-binding Activity and Transactivation Function
of Runx2--
To investigate whether FGF2-stimulated Runx2 expression
increases the binding of Runx2 to its DNA binding sites,
electrophoretic mobility shift assays were performed using a
radiolabeled oligonucleotide Runx2 probe that hybridizes to the Runx2
binding site (OSE2) in the promoter of mouse osteocalcin. Thus, C2C12
cells were cultured in the presence or absence of FGF2, and their
nuclear extracts were incubated with the probe. In untreated cells,
considerable Runx2 DNA-binding activity was observed but FGF2 treatment
strongly enhanced this (Fig.
2A, lanes 2 and
3). The binding complex is Runx2-specific,
because it could be competed out by adding a molar excess of unlabeled
wild type OSE2 oligonucleotides as competitors (Fig. 2A,
lanes 4 and 5) but was not affected by mutant
competitors (Fig. 2A, lanes 6 and 7).
Moreover, the binding complex was supershifted by a monoclonal antibody
specific for the C-terminal portion of Runx2 (24) (Fig. 2A,
lane 10). Runx2 bound specifically to the wild type probe
but not to the mutant OSE2 sequence (Fig. 2A, lanes
8 and 9).

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Fig. 2.
FGF/FGFR signaling enhances Runx2 DNA binding
activity as well as transactivation function. A,
sequence-specific Runx2 protein-DNA interactions at the mouse
osteocalcin (OC) OSE2 element. C2C12 cells were treated with
or without FGF2 for 12-24 h. Cells were harvested, and nuclear
extracts were prepared and incubated with oligonucleotides
corresponding to wild type (wt) or mutated OSE2 sites
(mut) from the OC gene. The specificity and the
identity of the complexes were shown by competition assays and
supershift assays, respectively. B, MC3T3-E1 cells
transiently transfected with wild type (wild) or mutated
OC promoter constructs (mut; carrying a mutation
in the Runx2 binding sites) were treated with or without FGF2. Reporter
activities were determined 16-24 h after transfection. C,
Runx2-deficient cells (Runx2 / ),
non-osteoblastic C2C12 cells, and osteoblastic MC3T3-E1 (MC)
cells were transfected with the p6xOSE2-Luc reporter vector. After
overnight culture in medium containing FBS at normal concentrations,
the medium was replaced with BSA-supplemented serum-free media with or
without FGF2, and the cells were cultured an additional 16-24 h. The
cells were then lysed, and the luciferase activities were determined.
D, Runx2 / cells, MC3T3-E1 cells, and C2C12
cells were transfected with the p6xOSE2-Luc reporter vector together
with vectors expressing the constitutively active FGFR2 mutant
(FR2Y340H) or empty vector as a control. Cells were cultured and
luciferase activities were determined. E, MC3T3-E1 cells
were transiently co-transfected with the Runx2 expression vector, which
expresses the p57 Runx2 product, or the empty expression vector (mock),
together with the p6xOSE2-Luc reporter vector. After overnight culture,
the cells were treated with or without FGF2. The cells were harvested
after 16-24 h, and luciferase activities were determined. The
luciferase activity was normalized by the protein concentrations in the
respective lysates.
|
|
We next asked whether FGF/FGFR signaling might also enhance
Runx2-mediated transactivation of downstream genes. A Runx2 binding site within the osteocalcin promoter is needed for the transactivation of the gene (30). Osteocalcin gene expression is induced by FGF2 (31),
and it is likely that the induction is mediated by Runx2 binding to the
osteocalcin gene promoter. We confirmed this by transfecting MC3T3-E1
cells with osteocalcin promoter constructs consisting of
208 to +23
bp of rat osteocalcin proximal promoter that contains a Runx2 binding
site, then incubating the cells with and without FGF2, after which
reporter activities were determined. FGF2 treatment increased the
reporter activity. When the cells were transfected with an osteocalcin
promoter in which the Runx2 site was mutagenized, the FGF-mediated
reporter activation was abolished (Fig. 2B).
To more accurately quantitate the transcriptional activity of Runx2
protein that is induced by FGF/FGFR signaling, we used a more sensitive
reporter system, namely, the p6xOSE2-luciferase reporter vector
(p6xOSE2-Luc). When the p6xOSE2-Luc reporter plasmid was transiently
transfected into osteoblastic cells (MC3T3-E1 cells) or
non-osteoblastic cells (C2C12), the reporter activity was enhanced
~4-fold by FGF2 treatment (Fig. 2C). When the cells were
co-transfected with p6xOSE2-Luc and the vector expressing the
constitutively active mutant of FGFR2, FR2Y340H, both cell lines had 3- to 4-fold increases in luciferase activity even in the absence of FGF2
(Fig. 2D). In the Runx2
/
calvaria cell line (24) (hereafter referred to as Runx2
/
cells), which
cannot generate endogenous Runx2, neither FGF2 treatment nor
overexpression of FR2Y340H increased the reporter activity, as
expected. MC3T3-E1 cells were co-transfected with the Runx2 expression
vector, which expresses the p57 Runx2 product, together with the
p6xOSE2-Luc reporter vector. The forced expression of Runx2 enhanced
the basal activity of p6xOSE2-Luc promoter and the activity was further stimulated by FGF2 treatment, however, the extent of the increase by
FGF2 treatment was not quite different by exogenous Runx2
overexpression (Fig. 2E).
The Protein Kinase C Pathway Plays a Crucial Role in
FGF/FGFR-stimulated Runx2 Expression--
We next asked
which signaling pathway is responsible for the FGF/FGFR-mediated
up-regulation of Runx2 expression. We first examined the involvement of
the PKC pathway, because PKC is known to be activated by the FGF/FGFR
signal (32, 13). Pharmacological down-modulation of PKC activity with
calphostin C, a general PKC inhibitor, dramatically reversed the
FGF2-induced increase in Runx2 mRNA levels (Fig.
3A). Another specific
inhibitor of PKC
,
,
, and
, GF109203X, had a similar
effect (Fig. 3B). In contrast, PD98059, an MEK1/2-specific
blocker, or SB203580, a p38-specific inhibitor, did not alter
FGF2-stimulated Runx2 mRNA expression (Fig. 3, C and
D). Thus, FGF-mediated up-regulation of Runx2 mRNA
expression occurs mainly through the activation of the PKC pathway.

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Fig. 3.
FGF2-induced Runx2 expression is regulated by
the protein kinase C (PKC) pathway but not by the MAPK pathway.
A and B, C2C12 cells were treated with FGF2 with
or without calphostin C (Cal C) (A) or GF109203X
(GF) pretreatment (B). The cells were harvested
3 h later, and Runx2 mRNA expression was determined by
Northern blot analysis. C and D, C2C12 cells were
treated with FGF2 with or without PD98059 (PD)
(C) or SB203580 (SB) (D) pretreatment.
The cells were harvested 3 h later, and Runx2 mRNA expression
was determined by Northern blot analysis. Me2SO was used as
vehicle. Ribosomal RNA (rRNA) is shown as control for
equivalent loading of RNA.
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FGF-mediated Increases in Runx2 Transcriptional Activity Correspond
with PKC-mediated Increases in Runx2 Protein Levels--
We examined
the effects of inhibiting the PKC pathway on the Runx2 DNA binding
activity in C2C12 cells. The inhibition of the PKC pathway by
calphostin C completely blocked the FGF2-induced DNA binding of Runx2
(Fig. 4A). The inhibition of
PKC activity with calphostin C almost completely abolished
FGF2-mediated stimulation of p6xOSE2-Luc activity (Fig. 4B).
Western blot analysis showed that both the increase in the Runx2
binding to OSE2 and the increase in the transcriptional activity of
Runx2 correlate quite well with Runx2 protein levels, which are clearly
regulated by the PKC pathway (Fig. 4C). Thus, the PKC
pathway mediates the FGF/FGFR signal that up-regulates the expression
and DNA binding and transactivation properties of Runx2.

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Fig. 4.
The PKC pathway plays a major role in the
FGF2 stimulation of Runx2 binding activity and transactivation
function. A, C2C12 cells were treated with FGF2 with or
without calphostin C (Cal C) pretreatment. The cells were
harvested 24 h later, and nuclear extracts were prepared.
Electrophoretic mobility shift assays were performed with nuclear
extracts and 32P-labeled OSE2 oligonucleotides. The
arrow indicates the Runx2 complex. B, cells
stably transfected with the p6xOSE2-Luc reporter were plated and
cultured in DMEM containing FBS. When cells reached confluence, the
cells were transferred to serum-free medium with FGF2 with or without
calphostin C (Cal C) pretreatment. The cells were harvested
16-24 h later, and luciferase activities were determined.
C, the same samples as in A were examined by
Western blot analysis using the anti-Runx2 antibody. The
arrow indicates the Runx2 protein.
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|
PKC
Mediates FGF2-stimulated Runx2 Expression--
Until
recently, at least 11 PKC isoforms have been identified (33), and
previous work has shown that FGF2 differentially activates different
set of isoforms in different cells (34, 35). PKC isoforms express in
osteoblastic cells are
,
,
,
, and
isoforms (36), and
those are also expressed in MC3T3-E1 cells that are used in this
experiment. To narrow down the range of PKC isoforms that may be
involved in Runx2 regulation by FGF2, we could rule out several PKC
isoforms by using isoform-selective PKC inhibitors. Because
FGF2-stimulated Runx2 expression was blocked by GF109203X (Fig.
3B), a specific inhibitor of PKC isoforms
,
, and
,
we can rule out the involvement of the other isoforms in the
regulation. The increase in Runx2 expression by FGF2 was significantly
blocked by rottlerin (a PKC
-specific inhibitor) but was not affected
by Go6976, a specific inhibitor of PKC
and
I (Fig.
5A). Consequently, PKC
would be a candidate for mediating the effect of FGF2. Furthermore,
rottlerin inhibited the activation of 6xOSE2-Luc promoter by FGF2 in a
dose-dependent manner up to 12 µM (Fig.
5B). To confirm further the involvement of PKC
isoform in
the Runx2 expression by FGF2, C2C12 cells were transiently transfected
with a dominant negative PKC
(PKC
-KR). The forced expression of
PKC
KR abolished FGF2-stimulated Runx2 expression (Fig.
5C). Consistently, transient cotransfection of PKC
KR
and p6xOSE2-Luc vectors into MC3T3-E1 cells also strongly
down-regulated FGF2-stimulated 6xOSE2-Luc promoter activity (Fig.
5D).

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Fig. 5.
The PKC isoform
plays a key role in the FGF2 induced Runx2 expression.
A, MC3T3-E1 cells were treated with FGF2 with or without 200 nM Go6976 (Go) or 3 µM rottlerin
(Rott) pretreatment. Me2SO (0.1%) was used as
vehicle for the reagents. B, C2C12 cells stably transfected
with the p6xOSE2-Luc reporter were plated and cultured in DMEM
containing 10% FBS. When cells reached confluence, the medium was
replaced with serum-free DMEM with or without rottlerin prior to FGF2
treatment. C, C2C12 cells were transfected with pcDNA3
(mock) or dominant negative PKC (PKC -KR), and then the cells were
treated with or without FGF2. D, MC3T3-E1 cells were
co-transfected with the PKC -KR expression vector or the empty
expression vector (mock), together with the p6xOSE2-Luc
reporter vector. For Northern blot analysis, the cells were harvested
after 3-h FGF2 treatment, and the consistent RNA loading was visualized
by hybridization with 18 S ribosomal RNA (18S) in the
respective blot (A and C). For the luciferase
assay, the cells were harvested after 16-24 h of FGF2 treatment, and
the luciferase activity was normalized by the protein concentrations in
the respective lysates (B and D).
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Post-translational Modification of Runx2 Protein by PKC Contributes
to FGF2-stimulated Runx2 Transcriptional Activity--
The
observations made so far strongly support the notion that the increase
in the transcriptional activity of Runx2 depends on increases in Runx2
protein levels resulting from FGF-induced PKC activation that
up-regulates Runx2 expression. We noted, however, that the
Western blot analysis revealed that protein levels of Runx2 were not
completely abolished by calphostin C treatment. In contrast, the
binding of Runx2 to OSE2 was completely inhibited by the blocker. In
other words, Runx2 protein level was still maintained comparable to
basal level in Western blot analysis but the Runx2 binding activity was
completely disappeared by the PKC blocker. This discrepancy suggests
that the PKC pathway may also be involved in a post-translational
modification of Runx2 protein that is needed for it to conduct its DNA
binding and/or transactivation activities. To investigate this, we used
Runx2
/
cells and introduced a Runx2-expression vector
together with the p6xOSE2-Luc reporter vector. The presence of the
Runx2-expressing vector increased the basal reporter activity by about
5-fold. FGF treatment of the doubly transfected cells stimulated the
reporter activity by 3-fold (i.e. 15-fold higher than the
basal reporter activity in cells transfected with the p6xOSE2-Luc
reporter vector only). Runx2
/
cells cannot express
endogenous Runx2 in response to FGF stimulation, and thus the increase
of Runx2-mediated reporter activity caused by adding FGF2 must be due
to the FGF2-mediated post-translational modification of the exogenously
introduced Runx2. That the PKC pathway mediates this post-translational
modification is shown by pretreating the cells with calphostin C. Calphostin C treatment had a little effect on the transactivating
activity of exogenously introduced Runx2 in the doubly transfected
cells, but it completely blocked the further increase in the reporter activity induced by FGF2 treatment (Fig.
6).

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Fig. 6.
The PKC pathway is also involved in the
post-translational modification of the Runx2 protein.
Runx2 / cells were transiently co-transfected with the
Runx2 expression vector, which expresses the p57 Runx2 product, or the
empty expression vector (mock), together with the
p6xOSE2-Luc reporter vector. After overnight culture, the cells were
treated with FGF2 with or without calphostin C (Cal C)
pretreatment. The cells were harvested after 16-24 h, and luciferase
activities were determined. Me2SO was used as
vehicle.
|
|
 |
DISCUSSION |
Runx2 Is a Downstream Target of FGF/FGFR
Signaling--
During the development of the vertebrate skeleton,
FGF/FGFR signaling appears to be closely involved in the determination of the shape and size of the skeleton (37). The importance of these
molecules in skeletal development is supported by the association of
several FGFR mutations with dominantly inherited human
skeletal disorders (9, 37, 38), as mutations in FGFR1,
FGFR2, and FGFR3 are linked to several
craniosynostosis related syndromes characterized by premature fusion of
the cranial sutures. These observations indicate that FGF/FGFR
signaling accelerates bone growth by inducing the rapid differentiation
of osteoprogenitor cells to bone cells. However, little is known about
the underlying molecular mechanisms and the downstream target genes of
FGF/FGFR signaling. In this matter, Zhou et al. (21)
recently provided an important clue when they showed that Runx2, an
essential transcription factor involved in osteoblast differentiation,
could be a downstream target of FGF/FGFR signaling. They revealed that
the introduction of the Pro-250
Arg mutant of
FGFR1, which is a constitutively active form of
FGFR1, resulted in craniosynostosis in the mouse and that
this was associated with an increase in Runx2 expression in the bone
tissue. They also showed that FGF8 treatment of bone cells stimulated
Runx2 expression. Our experiments reported here support
these observations, because we showed that Runx2 mRNA levels
increase upon FGF2 treatment or transfection of the cells with a vector
expressing the constitutively active FGFR2 mutant. Indeed,
our observations indicate that FGFs and FGFRs other than those studied
by Zhou et al. perform the same functions with regard to
Runx2 expression, because, like FGF8, FGF2 and FGF4 also stimulate Runx2 expression and, like the FGFR1 mutant, the
constitutively active FGFR2 mutant also increases Runx2
expression. Furthermore, FGF2 treatment induces Runx2 expression in
both bone and non-bone cells. These observations suggest that Runx2
could in fact be the general target of FGF/FGFR signaling. Thus,
although the different FGF ligands are specific for particular
receptors (40) and FGFR1 and FGFR2 play distinct differentiation- and
proliferation-related roles in the developing skull (41), it appears
that FGF2, FGF4, and FGF8 and FGFR1 and FGFR2 may play equivalent roles
in stimulating Runx2 expression.
More recently, Tsuji et al. (42) published the opposite
observation that FGF2 treatment transiently suppresses Runx2 expression in ROS17/2.8 cells. We show here, however, that FGF2 treatment up-regulates Runx2 mRNA levels in these cells. This discrepancy between their study and ours may be explained by differences in the
experimental conditions, particularly the length of FGF2
treatment and the presence of FBS in the medium. We have already shown
earlier that Runx2 expression is stimulated by BMP2 treatment but that the expression levels fluctuate after the immediate increase (24). This
may be due to the fact that there are multiple Runx2 binding sites in
the Runx2 promoter and Runx2 expression is stringently autoregulated by
its own protein (43). FGF2-stimulated Runx2 expression appears to be an
early event, because it peaks within 3 h of the initiation of
treatment, after which expression in the presence of continuous FGF2
treatment fluctuates (data not shown). Tsuji et al. (42)
treated their cells with FGF2 for 6-24 h and thus may not be
documenting the initial response, rather, they may be measuring the
fluctuations that arise from the auto-feedback regulation of the gene
product. Moreover, they did not exclude FBS from the medium when they
were treating the cells with FGF2. FBS contains many growth factors and
thus should be eliminated to clearly demonstrate the effect of adding
growth factors.
FGF Signaling Activates the PKC Pathway, Which Elevates
Runx2 Activity by Increasing Its Transcription--
Runx2 controls the
expression of osteoblast differentiation-related genes by binding to
OSE2 sites in the promoters of target genes (14). Although it has been
reported that transforming growth factor
1 and bone morphogenetic
protein 2 both induce Runx2 gene expression and stimulate the
binding of Runx2 to its recognition site (24, 23), it was unclear
whether FGF2-stimulated Runx2 expression is associated with an increase
in the DNA binding activity of Runx2. Our experiments reported here
show that FGF/FGFR signaling does increase Runx2 binding to the
Runx2-binding consensus sequence in the osteocalcin promoter and that
this activates the osteocalcin promoter. That mutation of the Runx2
binding site eliminated the FGF2-mediated increase in Runx2 DNA binding
and activation of the osteocalcin promoter strongly suggest that
FGF/FGFR-stimulated Runx2 activity results mainly from increases in
Runx2 transcription.
We next determined which signaling pathway is responsible for the
FGF2-mediated increase in Runx2 expression. There is a wealth of
information regarding the signals that stimulate Runx2 activity in
osteoblast differentiation, especially those transmitted by the
transforming growth factor
superfamily (24, 44, 45), but as yet,
nothing is known about the signal transduction mechanisms through which
FGF/FGFR controls the expression of Runx2. FGF binding to FGFR is known
to stimulate receptor dimerization, tyrosine phosphorylation, and the
activation of multiple signal transduction pathways, including those
involving Ras, mitogen-activated protein kinases (MAPKs), extracellular
signal-regulated kinases (Erks), src, and p38 MAPKs, phospholipase C,
and protein kinase C (PKC) (46-48). We used specific inhibitors to
investigate the role of some of these pathways in the
FGF2-mediated increase of Runx2 expression. We found that, although
FGF/FGFR signaling activates Erk1/2 and p38 MAPK in osteoblasts
(10-12), inhibition of MEK1/2 or p38 MAPK did not block FGF2
stimulation of Runx2 expression in osteoblast-like MC3T3-E1 or
non-osteoblast C2C12 cells (Fig. 3, C and D).
However, the inhibition of the PKC pathway by selective and potent PKC
inhibitors (calphostin C and GF109203X) and upstream phospholipase
C-
inhibitor (U73122, data not shown) dramatically decreased
FGF2-stimulated Runx2 mRNA levels in MC3T3-E1 and C2C12 cells (Fig.
3, A and B). Because the Runx2 protein levels in
FGF2-stimulated calphostin C-treated cells correlated fairly well with
the mRNA levels (Fig. 4C), it appears that the PKC
pathway activated by the FGF/FGFR signal acts to modulate Runx2
activity by up-regulating its transcription.
The PKCs have been classified into three groups based upon their
ability to be activated by Ca2+ and diacylglycerol (DAG).
The classic PKCs are activated by both Ca2+ and DAG and
include the
,
I,
II, and
isoforms. The
Ca2+-independent but DAG-dependent isoforms
(
,
,
, and
) comprise the novel PKCs. Finally, the atypical
PKCs,
,
/
, and µ, are both Ca2+- and
DAG-independent. Thus, it is difficult to reveal the role of each PKC
isoform in this regulation. However, two lines of evidences point to
the involvement of PKC
in FGF2-stimulated Runx2 regulation. One is
that rottlerin, a specific inhibitor of the
subtype, suppressed the
FGF2-stimulated Runx2 expression and its transcriptional
activity (Fig. 5, A and B). The other is that the
overexpression of the inactivated PKC
in C2C12 or MC3T3-E1 cells,
which express PKC
subtype (49), abolished the increase in Runx2
expression and 6xOSE2-Luc promoter activity by FGF2 (Fig. 5,
C and D). Recent studies showed that FGF2
stimulated PKC
activation in neuronal cells and pituitary tumor
cells (50-52). Collectively, these results strongly indicate that
PKC
is a key mediator of FGF2-stimulated Runx2 regulation. Although
we've shown the role of PKC
in FGF2-stimulated Runx2 expression, we
could not completely rule out the involvement of other PKC isoforms, because the treatment of rottlerin or the transfection of PKC
-KR could not completely reversed the FGF2-stimulated Runx2 transcriptional activity (Fig. 5, B and D).
PKC Activation by FGF/FGFR Signaling Also Stimulates
Runx2 Activity by a Post-translational Mechanism--
Several lines of
evidence indicate that the stimulation of the Runx2 expression would be
the main component of the FGF2-stimulated Runx2 transcriptional
activity; first, the activation of FGF/FGFR signaling could not
stimulated Runx2 transcriptional activity in Runx2
/
cells (Fig. 2, C and D). Second,
forced expression of Runx2 is sufficient to increase Runx2
transcriptional activity even in the absence of FGF2 treatment (Fig.
2E). Third, the mRNA or protein induction level by FGF2
was quite well correlated with Runx2 transcriptional activity (Fig. 4).
However, there was a minor discrepancy between the protein level of
Runx2 and its binding activity, because, although the PKC inhibitor
reversed the FGF2-stimulated Runx2 protein level only to its basal
level, it completely abolished the DNA binding activity and
transactivating function of the Runx2 protein (Fig. 4, A and
C). Despite this, there was a good correlation between Runx2
mRNA and protein levels. Thus, there must be an additional
regulatory mechanism through which the PKC pathway controls the
activity of Runx2 protein. To confirm this notion, we used
Runx2
/
cells, because these cells cannot express
endogenous Runx2 protein in response to FGF2 signaling. FGF2 treatment
up-regulated the transactivating activity of the exogenously
overexpressed Runx2 and the PKC inhibitor reversed this (Fig. 6). Thus,
although the PKC pathway up-regulates Runx2 activity largely by
elevating Runx2 expression, it also increases the transactivating
function of Runx2 protein by an as yet undetermined post-translational modification.
Thus, our data show that the PKC pathway is involved in
regulating both FGF/FGFR-stimulated Runx2 expression and the
transcriptional activity of the Runx2 protein. Furthermore, MAPK
pathways are not involved in Runx2 expression. However, we cannot
completely rule out the possibility that MAPKs participate in the
post-translational regulation of Runx2. Two reports indicate that the
MAPK pathway contributes to the post-translational regulation of Runx2
that is involved in the extracellular matrix-stimulated induction of alkaline phosphatase, an osteoblast differentiation-related gene. This
is based on the observation that the specific inhibition of the ERK
pathway selectively and rapidly blocks the extracellular matrix-stimulated activity of the osteocalcin promoter that depends on
the post-translational regulation of the Runx2 protein (53, 54). Thus,
the contribution of the MAPK pathway and cross-talk with PKC pathway in
the post-translational regulation of the Runx2 protein resulting from
FGF/FGFR signaling should be further investigated. Moreover, Runx2 is a
nuclear matrix protein (55) and is known to interact with many
transcription factors such as Cbf-
, AP1, C/EBPs, and Smads (24, 25,
39, 56). Understanding the contribution of these transcription factors
to FGF/FGFR signaling will be of great importance in elucidating the
general mechanism through which Runx2 is regulated by FGF.
 |
ACKNOWLEDGEMENTS |
The FGFR mutant found in patients with
Crouzon syndrome was generously provided by Dr. Daniel Donoghue,
Department of Chemistry and Biochemistry, University of California, San
Diego, CA. OC208-CAT and OC208-CAT Runx mut reporter constructs and
Runx2-type II expression vector were provided by Drs. Gary S. Stein and Jane B. Lian, Department of Cell Biology,
University of Massachusetts Medical School, Worcester, MA. Dominant
negative PKC
expression vector (PKC
-KR) was generously provided
by Dr. Jae-Won Soh, Department of Biochemistry & Molecular Biophysics and Herbert Irving Comprehensive Cancer Center,
Columbia University, NY.
 |
FOOTNOTES |
*
This work was supported by Grant KRF-99-041-F00281 from the
Korea Research Foundation and General Research Grant KOSEF
1999-2-209-008-5 from the Korea Science and Engineering Foundation.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: Dept. of Biochemistry,
School of Dentistry, Kyungpook National University, 101 Dong In-Dong,
Jung-Gu, Daegu 700-422, Korea. Tel.: 82-53-420-6815; Fax:
82-53-421-1417; E-mail: hmryoo@knu.ac.kr.
Published, JBC Papers in Press, October 25, 2002, DOI 10.1074/jbc.M203750200
 |
ABBREVIATIONS |
The abbreviations used are:
FGF, fibroblast
growth factor;
FGFR, FGF receptor;
MAPK, mitogen-activated protein
kinase;
Erk, extracellular signal-regulated kinase;
MEK1, MAPK/Erk
kinase;
PKC, protein kinase C;
Runx2, Runt-related transcription factor
2;
BSA, bovine serum albumin;
-MEM,
-minimal essential medium;
DMEM, Dulbecco's modified Eagle's medium;
FBS, fetal bovine
serum;
PBS, phosphate-buffered saline;
MOPS, 3-[morpholino]propanesulfonic acid;
OSE2, osteoblast-specific core
binding sequences;
DAG, diacylglycerol.
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