Evidence for a Role of p38 MAP Kinase in Expression of
Alkaline Phosphatase During Osteoblastic Cell Differentiation
A. SUZUKI,1,2
J. GUICHEUX,1
G. PALMER,1
Y. MIURA,2
Y. OISO,2
J.-P. BONJOUR,1
and
J. CAVERZASIO1
1
Division of Bone Diseases, Department of Internal Medicine, University Hospital of Geneva, Geneva, Switzerland
2
First Department of Internal Medicine, Nagoya University School of Medicine, Nagoya, Japan
In the present study, we investigate the implication of the
mitogen-activated protein kinases (MAPKs) Erk, p38, and
JNK in mediating the effect of fetal calf serum (FCS) on
the differentiation of MC3T3-E1 osteoblast-like cells. Erk
is stimulated by FCS in proliferating, early-differentiating,
as well as in mature cells. Activation of p38 by FCS is
not detected in proliferating cells but is observed as the
cells differentiate. JNK is activated in response to FCS
throughout the entire differentiation process, but a maximal
stimulation is observed in early differentiating cells.
The roles of Erk and p38 pathways in mediating
MC3T3-E1 cell differentiation was determined using specific
inhibitors such as U0126 and SB203580, respectively.
These experiments confirmed that the Erk pathway is
essential for mediating cell proliferation in response to
FCS, but indicated that this MAP kinase has little effect in
regulating the differentiation of MC3T3-E1 cells. In contrast,
p38 only marginally influenced proliferation, but
appeared to be critical for the control of alkaline phosphatase
(ALP) expression in differentiating cells. Finally,
results obtained with high doses of SB203580, which also
affected JNK activity, suggest that p38 and/or JNK are
probably also involved in the control of type 1 collagen and
osteocalcin expression in differentiating cells. The data
indicate that MAPKs regulate different stages of
MC3T3-E1 cell development in response to FCS. Distinct
MAPK pathways seem to independently modulate osteoblastic
cell proliferation and differentiation, with Erk
playing an essential role in cell replication, whereas p38 is
involved in the regulation of ALP expression during osteoblastic
cell differentiation. JNK is also probably involved
in the regulation of osteoblastic cell differentiation,
but its precise role requires further investigation. (Bone
30:91­98; 2002)  2002 by Elsevier Science Inc. All
rights reserved.
Key Words: Osteoblast; Erk; p38 mitogen-activated protein
(MAP) kinase; JNK; Proliferation; Differentiation.
Introduction
The differentiation of preosteoblasts is dependent upon the temporal
regulation of multiple interacting signaling pathways. Recent
discoveries of transcription factors that control either bone
development or the expression of bone phenotypic markers have
significantly improved our understanding of molecular mechanisms
supporting osteoblast growth and differentiation.20
Upstream
cellular mechanisms responsible for either the expression
or the activation of these factors remain, however, largely unknown.
In particular, the role of mitogen-activated protein kinases
(MAPKs) in bone forming cells is poorly understood.
Three major subfamilies of structurally related MAPKs have
been identified in mammalian cells: the extracellular signalregulated
kinases (Erks); the c-Jun N-terminal kinases (JNKs);
and the p38 MAP kinases (p38s). MAPKs are proline-directed
serine-threonine kinases that have important functions as mediators
of cellular responses to a variety of extracellular stimu-
li.3,24,31
Erks are characteristically activated by various growth
factors and by phorbol esters. Members of the JNK and p38
subfamilies are strongly activated in response to stress stimuli
(ultraviolet radiation, heat shock, and hyperosmolarity18,26,30
),
and thus proinflammatory cytokines, and have been given the
name stress-activated protein kinases (SAPKs). Whereas the Erk
pathway is usually associated with cell proliferation and protection
from apoptosis, JNK and p38 can promote apoptosis in many
systems.10,15,16,39
Recent data suggest that, in addition to its
effect on apoptosis, the p38 MAPK pathway might also be
involved in the differentiation of neuronal cells12,27
and adipocytes.9
In osteoblast-like cells, activation of Erks has been reported in
response to several growth factors including mitogens acting
either through receptor tyrosine kinases (RTKs) such as basic
fibroblast growth factor (bFGF),5,36
platelet-derived growth factor
(PDGF),5,42
insulin-like growth factor-1 (IGF-1),14
epidermal
growth factor (EGF),25
or G-protein-coupled receptors (GPCRs)
like prostaglandin F2 ,11
sphingosine 1-phosphate,17
endothelin-
1,13
and fluoride.4,33
Activation of Erks has also been reported in
response to cross-linking of collagen with integrins,37
17 -
estradiol,8
interleukin-6,28
and hypoxia.25
As reported in other
cell systems, activation of Erks by growth factors in osteoblastlike
cells is associated with enhanced cell proliferation. However,
recent data have suggested that this MAPK might also be
involved in the regulation of bone cell differentiation.13,37,38
Little information has been provided on the regulation and role of
the JNK and p38 pathways in bone-forming cells. Recently, we
found that the p38 pathway mediates the stimulation of alkaline
Address for correspondence and reprints: Dr. Joseph Caverzasio, Division
of Bone Diseases, Department of Internal Medicine, University
Hospital of Geneva, 1211 Geneva 14, Switzerland. E-mail: Joseph.
Caverzasio@medecine.unige.ch
Bone Vol. 30, No. 1
January 2002:91­98
91 2002 by Elsevier Science Inc. 8756-3282/02/$22.00
All rights reserved. PII S8756-3282(01)00660-3
phosphatase (ALP) activity induced by activation of a Gi-protein-coupled
receptor agonist in osteoblast-like cells,34,35
suggesting
that this MAP kinase could be involved in the differentiation
of bone-forming cells. To further determine the role of
MAP kinases in the differentiation of osteoblastic cells, we used
murine calvarial-derived MC3T3-E1 cells, which is an in vitro
model of osteoblast development. These cells express distinct
proliferative and differentiating stages. During the initial phase
of culture (days 1­8), MC3T3-E1 cells actively replicate, but do
not express, ALP. By days 6­9, cells undergo growth arrest and
start to express osteoblastic functions, including production of
ALP, processing of procollagens and incremental deposition of a
collagenous extracellular matrix, as well as secretion of osteo-
calcin.32
In the present work, we sought to determine which
MAP kinases are activated by fetal calf serum (FCS), an essential
growth factor supplement for the normal in vitro differentiation
of these cells,41
and analyzed the role of MAP kinases in
osteoblast differentiation using specific inhibitors.
Materials and Methods
Chemicals
Cell culture plasticwares were purchased from Falcon (BectonDickinson,
Franklin Lakes, NJ) and Corning-Costar (Integra
Biosciences, Switzerland). Fetal calf serum (FCS), glutamine,
antibiotics, and trypsin/ethylene-diamine tetraacetic acid
(EDTA) were from Gibco (Life Technologies, Basel, Switzerland).
-modified essential medium ( MEM) was purchased
from Amimed (Bioconcept, Allschwill, Switzerland). 4-(4fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)
imidazole
(SB203580) and type I collagenase were from Sigma
Co. (St. Louis, MO). 1,4-diamino-2,3-dicyano-1,4-bis(oaminophenylmercaptobutadiene)
(U0126) was obtained from
Tocris (Baldwin, MO). All reagents for sodium dodecylsulfatepolyacrylamide
gel electrophoresis (SDS-PAGE) were from BioRad
(Munich, Germany). GeneScreen membranes were from
DuPont de Nemours (Brussels, Belgium). [ -32
P]deoxy-CTP and
[ -32
P]ATP were obtained from Amersham Pharmacia Biotech
(Little Chalfont, UK). All other chemicals were from standard
laboratory suppliers and were of the highest purity available.
Antibodies
Antibodies against ERK2 (sc-154-G), p-JNK (agarose-conjugated,
sc-6254), c-Jun (D11:sc-7481), p-c-Jun (KM-1: sc-822), JNK
(sc-571), and p38 (SC-535-G) were obtained from Santa Cruz
Biotechnology, Inc. (Santa Cruz, CA). Antibodies against p-Erk
(agarose-conjugated, #9109) and p-p38 (agarose-conjugated,
#9219) were purchased from New England Biolabs (Beverly,
MA). The antibody against MAPKAP-K2 was from StressGen
(Victoria, BC, Canada).
Cell Culture
Mouse calvaria-derived MC3T3-E1 cells were cultured in
-minimal essential medium ( MEM) containing 10% FCS
(vol/vol), 0.5% nonessential amino acids (vol/vol), 100 IU/mL
penicillin, and 100 g/mL streptomycin. They were seeded at
10,000 cells/cm2
and cultures were maintained at 37°C in a
humidified atmosphere of 5% CO2-95% air and the culture
medium was changed every 2­3 days. In the subclone of
MC3T3-E1 cells used in this study, cells cultured in MEM and
10% FCS proliferated rapidly during 6­7 days with a decline in
growth between days 8 and 10. During this time period, cells
began to express bone markers such as ALP, type I collagen
(Coll type I), and osteocalcin (OC). Maximal expression of these
markers was reached after approximately 25 days. In experiments
aimed at testing the effect of the MAPK inhibitors, agents
were added 1 h prior and with each medium change (every 2­3
days).
Analysis of Markers of Bone Cell Differentiation
Cells cultured in six well tissue culture clusters for various time
periods were harvested in 1 mL of 0.2% Nonidet P-40 and the
cell suspension was disrupted by sonication. After centrifugation
at 1500g for 5 min, ALP activity was measured in the supernatant
by the method of Lowry et al.22
Collagen synthesis was determined by measuring the 4-hydroxyproline
content in the cell layer. Briefly, cells were hydrolyzed
in 6N HCl for 16 h at 116°C. After lyophilization and
reconstitution of the lysate in distilled water, the amount of
4-hydroxyproline was determined by spectrophotometry at 550
nm as previously described.1
Osteocalcin released in the medium was measured by radioimmunoassay
using a goat anti-mouse osteocalcin antibody and
a donkey anti-goat secondary antibody (Biomedical Technologies,
Inc., Stoughton, MA) as previously described.6
DNA content was measured as described by Burton2
with calf
thymus DNA as standard and cell replication by cell counting
(Coulter counter).
The amount of protein was determined using the Pierce
Coomassie Plus assay reagent (Pierce, Rockford, IL).
RNA Extraction and Northern Blotting
Cells were cultured in 25 cm2
flasks for various times and total
RNA was isolated using the TriPure Isolation Reagent from
Roche Molecular Biochemicals (Rotkreuz, Switzerland). Cell
layers were scraped into 1 mL of TriPure Reagent and homogenized
with a pipette. Cell lysates were then cleared by centrifugation
before addition of 0.2 mL chloroform. After mixing, the
samples were separated into three phases by centrifugation.
Finally, RNA contained in the upper aqueous phase was precipitated
with 0.5 mL isopropanol, washed with 75% ethanol,
air-dried, and resuspended in water.
For northern blotting analyses, 10 g of total RNA were
denatured in glyoxal and separated by electrophoresis on a 1.2%
agarose gel. RNA was transferred to GeneScreen membranes by
capillary transfer. Membranes were ultraviolet cross-linked and
stained with methylene blue to control equal sample loading and
RNA integrity. The membranes were then hybridized to the rat
ALP complementary deoxyribonucleic acid (cDNA) probe labeled
with 50 Ci of [ -32
P]deoxy-CTP by random priming
(Megaprime DNA labeling system, Amersham Life Sciences).
The rat ALP cDNA probe, a 2.5 kb EcoRI restriction fragment
purified from the pBS plasmid, was provided by Dr. G. Rodan
(Merck Sharp and Dohme Research Laboratories, West Point,
PA). Prehybridization (2 h) and hybridization (3 h) were performed
in QuickHyb hybridization solution (Stratagene, La Jolla,
CA), which was supplemented with 100 g/mL salmon sperm
DNA. Membranes were washed twice for 15 min in 2 standard
saline citrate (SSC) and 0.1% sodium dodecylsulfate (SDS) at
25°C and for 10­30 min at 65°C in 0.1 SSC, 0.1% SDS before
exposition to Kodak X-Omat AR films for 18­72 h at 80°C.
Western Blotting and Kinase Assay
Cells cultured in 75 cm2
flasks were exposed to fresh culture
medium for various time periods and frozen in liquid nitrogen.
Whole cell lysates were obtained by solubilizing the cells at 4°C
92 A. Suzuki et al. Bone Vol. 30, No. 1
p38 MAP kinase and osteoblastic cell differentiation January 2002:91­98
in 3 mL lysis of buffer A containing 50 mmol/L Tris (pH 7.4),
150 mmol/L NaCl, 1 mmol/L phenylmethylsulfonylfluoride, 10
g/mL aprotinin, 10 g/mL leupeptin, 2 mmol/L Na3VO4, 0.01
mol/L calyculin A, 0.1 mol/L mycrocystin LR, 1% NP-40,
1% sodium deoxycholate, and 0.1% SDS. The cell lysates were
cleared by centrifugation at 6000 rpm, 4°C, for 30 min. For
western blotting analysis, an aliquot of the supernatant was
diluted with an equal amount of 2 reducing sample buffer
consisting of 125 mmol/L Tris (pH 6.8), 20% glycerol, 4% SDS,
200 mmol/L dithiothreitol, and 0.025% bromophenol blue. For
kinase assays, similar amounts of protein lysate were incubated
with the appropriate agarose-conjugated antiphospho-MAPK antibody
for 18 h at 4°C. Immunoprecipitates were washed two
times in buffer A and two times in kinase buffer containing 50
mmol/L Tris (pH 7.4), 25 mmol/L -glycerophosphate, 20
mmol/L MgCl2, 1.0 mmol/L dithiotreitol, 10 mol/L 32
P-ATP
(50 Ci/mL), and 2.5 g of either GST-Elk1 (New England
Biolabs) for Erk assay; GST-ATF2 (Santa Cruz Biotechnology)
for p38 assay, GST-c-Jun (Biomol Research Laboratories, Campus
Drive, PA) for JNK assay, or recombinant HSP27 (StressGen,
Victoria, BC, Canada) for MAPKAP-K2 assay, and incubated
for 30 min at 30°C. The reaction was stopped by addition
of 2 reducing buffer. The samples were heated at 70°C for 30
min, fractionated by reducing SDS-PAGE on 6%­15% acrylamide
gradient gels, and transferred to Immobilon P membranes
(Millipore Corp., Bedford, MA). Immobilon P membranes were
finally exposed for autoradiography at 80°C. Immunoblotting
was performed as described previously with appropriate primary
antibodies and horseradish peroxidase-conjugated secondary an-
tibodies.4
Immunoreactive bands and biotinylated molecular
weight standards were visualized by ECL (Amersham Pharmacia
Biotech).
Statistical Analysis
Results are expressed as the mean SEM. Comparative studies
of means were performed using one-way analysis of variance
followed by a post hoc test (Fisher's projected least significant
difference) with a statistical significance at p 0.05.
Results
Growth factors contained in FCS have been shown to play a
critical role in the growth and differentiation of MC3T3-E1
cells.41
It is well documented that many growth factors can lead
to the stimulation of different MAP kinases. We therefore investigated
the effects of FCS on activation of the Erk, p38, and JNK
pathways in these cells. Data shown in Figure 1 indicate that
addition of fresh culture medium containing 10% FCS in early
differentiating (day 10) MC3T3-E1 cells induces activation of
the three types of MAPKs. Activation of the Erk pathway was
rapid and already maximal after 5 min. The response then
gradually decreased, although slight stimulation was still detected
after 3 h (Figure 1A, upper panel). The kinetics of p38 and
JNK activation by FCS were quite different from that observed
from Erk. The stimulation of p38 was apparent after 15 min with
a maximal effect detected at 1 h. The response then disappeared
rapidly (Figure 1A, middle panel). Activation of the JNK pathway
was also detected after 15 min with a maximal effect at 1 h,
but the JNK response was more pronounced compared with p38
(Figure 1A, lower panel). Analysis of MAPK activation in
response to various doses of FCS in MEM medium indicates
that there was no activation of the Erk, p38, and JNK pathways
when medium without FCS was added (Figure 1B). In this study,
we observed a difference in Erk activation by various doses of
FCS as compared with the stimulation of the two other MAPK
pathways. A low concentration of FCS (1%) was sufficient to
slightly enhance the activity of Erk, but not that of p38 and JNK.
In addition, Erk was maximally stimulated by 5% FCS, whereas
this concentration of FCS was not sufficient for maximal activation
of p38 and JNK (Figure 1B). This observation suggests
that the growth factors contained in FCS differentially stimulate
these three types of MAPKs in osteoblast-like cells.
To determine the respective role of these MAPKs in controlling
the proliferation and differentiation of osteoblast-like cells,
we analyzed MAPK activation by FCS at various stages of
MC3T3-E1 cell differentiation. Data shown in Figure 2 indicate
that activation of the Erk pathway by FCS was detected at all
stages of MC3T3-E1 cell development. In contrast, the p38 MAP
kinase pathway was not activated in proliferating cells. It was,
however, maximally stimulated in early-differentiating cells and
marginally activated in late-differentiating MC3T3-E1 cells (Figure
2, middle panel). A stimulation of the JNK pathway by FCS
was already detected during proliferation. The response was,
however, maximal in early-differentiating and still present in
late-differentiating cells (Figure 2, lower panel). The amounts of
the various MAPKs was relatively constant throughout the culture
periods except for JNK2 expression, which was increased by
2.4- and 4.1-fold in differentiating cells at days 11 and 18,
respectively, as compared with levels in proliferating cells (Figure
2, lower panel).
The observation that the three types of MAPKs (Erk, p38, and
JNK) are activated by FCS in differentiating MC3T3-E1 cells
suggested a possible role(s) for these signaling pathways in the
establishment of the osteoblastic phenotype. To test the role of
Erk, we used the specific MEK inhibitor, U0126. A dose of 1
mol/L was sufficient to block almost completely the stimulation
of Erk induced by FCS in differentiating MC3T3-E1 cells
without influencing the activation of p38 and JNK (Figure 3A).
Figure 1. Activation of Erk, p38, and JNK in MC3T3-E1 cells after
addition of fresh culture medium. MC3T3-E1 cells were cultured for 10
days in complete MEM medium containing 10% FCS. Then, cells were
either kept in the same medium or changed with fresh MEM medium
containing 10% FCS for various incubation times (A) or with MEM
culture medium containing different amounts of FCS (B) for 5 min (Erk
determination) or 1 h (p38 and JNK determinations). At the end of the
incubation period, cells were rapidly frozen in liquid nitrogen before lysis
at 4°C. Lysates were then immunoprecipitated with specific antiphosphoMAPK
antibodies. Immune complexes were incubated with the appropriate
substrate and [ -32
P]ATP in phosphorylation buffer as described in
Materials and Methods. Following in vitro phosphorylation, proteins
were heated in SDS sample buffer, subjected to SDS-PAGE electrophoresis,
transferred to Immobilon P membranes, and autoradiographed.
The upper panel of each pair of images is an autoradiogram of the
phosphorylated kinase substrate, whereas the lower panel is a western
blot of the corresponding kinase protein.
93Bone Vol. 30, No. 1 A. Suzuki et al.
January 2002:91­98 p38 MAP kinase and osteoblastic cell differentiation
This dose of U0126 was also sufficient to completely block the
increase in DNA content observed between day 8, when cells
reach the end of their proliferative phase, and day 11, when cells
stop proliferating (day 8: vehicle, 80.1 1.0; day 11, vehicle,
101.4 2.0; day 11: U0126, 84.5 1.4 g DNA/well). This
latter observation confirms that the Erk pathway is involved in
the control of cell proliferation in response to growth factors in
osteoblastic cells. Associated with the selective inhibition of Erk
activation and cell proliferation by U0126, we found that the
activity of ALP and the deposition of type 1 collagen were, in
general, slightly more elevated in differentiating cells treated
with this inhibitor as compared with controls (Figure 3B,C). A
very small, nonsignificant decrease in the stimulation of osteocalcin
secretion was deteced in early-differentiating cells treated
with the inhibitor, but this effect was no longer present in
late-differentiating cells (Figure 3D).
The role of the p38 pathway was investigated with the
SB203580 inhibitor.21
Data shown in Figure 4 indicate that 10
mol/L SB203580 almost completely blocked expression of
ALP activity in differentiating MC3T3-E1 cells (Figure 4A).
This effect was observed in the presence of a slightly higher
proliferation and a slight decrease in the protein content (Table
1), suggesting that this inhibitor had no cytotoxic effect. This was
further confirmed by the nearly complete reversibility of inhibition
when SB203580 was removed (Figure 4B). Associated with
its effect on ALP activity, SB203580 also blocked the increase in
ALP mRNA expression induced by 10% FCS (Figure 3C),
Figure 2. Activation of Erk, p38, and JNK after addition of fresh culture
medium as a function of time in culture. MC3T3-E1 cells were cultured
for the indicated number of days in complete MEM medium containing
10% FCS. Then, at various time intervals, fresh MEM medium containing
10% FCS was added for 15 min and 1 h. At the end of the
incubation periods, cells were rapidly frozen in liquid nitrogen before
lysis at 4°C. Lysates were then immunoprecipitated with specific antiphospho-MAPK
antibodies. In vitro kinase assays and western blotting
analyses were performed as described in the legend to Figure 1.
Figure 3. Effect of the MEK inhibitor, U0126, on activation of Erk, p38,
and JNK and on MC3T3-E1 cell differentiation. (A) MC3T3-E1 cells
were cultured for 8 days in complete MEM medium containing 10%
FCS. Then, cells were preincubated with various concentrations of
U0126 for 1 h before exposure to fresh culture medium containing 10%
FCS for 5 min (Erk determination) or 1 h (p38 and JNK determinations).
(B)­(D) To analyze the effect of U0126 on cell differentiation, cells were
treated with 1 mol/L of U0126 (hatched bars) or its vehicle (open bars)
as described in Materials and Methods. Expression of the osteoblastic
markers alkaline phosphatase (ALP [100% 76.4 nmol/min per milligram])
(B), type I collagen (Coll I [100% 9.53 g/mg]) (C), and
osteocalcin (OC [100% 0.68 ng/mg]) (D) was measured after various
times. In (B)­(D), values are the mean SEM of four or five determinations.
Each determination was corrected by the amount of protein and
expressed in percent of the maximal value recorded at day 25. *p 0.01
compared with the respective time control.
Figure 4. Effect of the p38 MAPK inhibitor, SB203580, on alkaline
phosphatase enzyme activity and mRNA expression in MC3T3-E1 cells.
(A) MC3T3-E1 cells were cultured for 4 days and exposed to either 10
mol/L SB203580 (filled circles) or its vehicle (open circles) for 8 days
as described in Materials and Methods. Then, the inhibitor was either
kept in the culture medium (filled circles) or removed (open triangles) for
various times before the determination of ALP activity. (C) The influence
of SB203580 (SB) on ALP mRNA expression was studied by northern
blotting using total RNAs (10 g) obtained from MC3T3-E1 cells
preincubated with either 10 mol/L SB203580 (SB) or its vehicle and
exposed to fresh culture medium containing 10% FCS for various times.
A picture of the methylene blue-colored membrane (Mbne) indicating the
amount of 28S rRNAs is shown below the blot. In (A) and (B), values are
the mean SEM of four determinations of a representative experiment.
p 0.01 compared with vehicle.
94 A. Suzuki et al. Bone Vol. 30, No. 1
p38 MAP kinase and osteoblastic cell differentiation January 2002:91­98
suggesting that the increase in p38-induced ALP expression in
differentiating cells occurs at the mRNA level.
SB203580 was reported to inhibit not only p38 but also
particular JNK isoforms.7
We thus verified whether there was a
good correlation between p38 inhibition in the cells and the
decrease in ALP activity in response to various doses of
SB203580. Data shown in Figure 5 indicate that there was
effectively a good relationship between the doses of SB203580
that inhibit FCS-induced expression of ALP activity, on the one
hand (Figure 5A), and those that decrease the activity of MAPKAP-K2,
on the other (Figure 5B). MAPKAP-K2 is a selective
and physiologic downstream effector of p38 and has been used as
a read-out to monitor cellular p38 activity.23
This observation
therefore strongly correlates p38 activity with ALP expression in
differentiating osteoblasts. In contrast, there was no apparent
relationship between changes in ALP expression in the presence
of low doses (1­10 mol/L) of SB203580 and inhibition of JNK
activity assessed by measuring the level of phosphorylated c-Jun
(Figure 5A and Figure 6B). However, we observed that Coll
type I deposition or OC secretion were inhibited only by higher
doses (10­30 mol/L) of SB203580. These alterations correlated
with a decrease in JNK activity (Figure 6), suggesting that
this MAPK may also be involved in controlling the expression of
osteoblastic differentiation markers other than ALP, and thus
may also play a role in osteoblastic differentiation. This relationship,
however, cannot be investigated at present because of a lack
of reagent for JNK.
Discussion
The results of the present study strongly suggest that the MAPKs
Erk and p38 are involved in the control of osteoblast proliferation
and differentiation. As mentioned earlier, the role of JNK could
not be investigated because of a lack of reagent. Activation of
these signaling pathways in differentiating MC3T3-E1 cells after
addition of fresh culture medium containing 10% FCS is mediated
by factors contained in the serum, because no stimulation of
these MAPKs was detected when fresh culture medium containing
no FCS was used (Figure 1, right panel). The serum factors
responsible for the stimulation of these MAPKs in osteoblastic
cells are not known, but likely involve ligands for different
types of receptors such as RTKs, GPCRs, and possibly cytokine
receptors.
Kinetic analysis, as well as the dose response to FCS, suggest
the implication of different signaling complexes for activation of
the three types of MAPKs by serum growth factors (Figure 1, left
panel). We observed that activation of p38 and JNK was significantly
delayed compared with the rapid stimulation of Erk by
FCS (Figure 1A). This observation and recent data indicating that
Table 1. Effect of SB203580 on MC3T3-E1 cell growth and alkaline phosphatase activity
Time
(days)
DNA content ( g/well) Protein content (mg/well)
Alkaline phosphatase activity
(nmol/mL per minute)
Veh SB Veh SB Veh SB
4 21.4 0.4 -- 0.20 0.01 -- 13.7 6.7 --
11 46.6 1.7 50.2 1.9a
0.76 0.01 0.76 0.01 203.7 7.7 53.3 0.8b
18 41.7 1.3 50.2 0.3a
1.08 0.01 0.93 0.02a
518.9 22 102.5 3.0b
25 42.0 1.1 46.6 1.3 1.27 0.01 1.15 0.01a
611.7 4.0 101.7 7.5b
MC3T3-E1 cells were cultured for 4 days before exposure to 10 mol/L SB203580 (SB) or its vehicle (Veh). At various timepoints,
cells were harvested for the determination of DNA and protein content as well as alkaline phosphatase activity. Each value represents
mean SEM of four determinations of a representative experiment.
a
p 0.01 compared with vehicle.
b
p 0.001 compared with vehicle.
Figure 5. Dose-dependent effect of SB203580 on alkaline phosphatase
and p38 MAPK activities. (A) MC3T3-E1 cells were cultured for 4 days
before exposure to various concentrations of SB203580 (hatched bars) or
its vehicle (open bars) for 7 days and ALP activity was determined as
described in Materials and Methods. Values are the mean SEM of four
determinations of a representative experiment. *p 0.01 compared with
vehicle. (B) MC3T3-E1 cells cultured for 10 days were preincubated
with various concentrations of SB203580 for 1 h. Then, fresh MEM
10% FCS culture medium containing either SB203580 or its vehicle were
added for a further 60 min and cells were rapidly frozen in liquid nitrogen
before lysis at 4°C. Lysates were then immunoprecipitated with a specific
anti-MAPKAP-K2 antibody and in vitro MAPKAP-K2 activity was
assessed by phosphorylation of HSP27 as described in Materials and
Methods. Changes in HSP27 phosphorylation content were quantified by
densitometry (OD).
95Bone Vol. 30, No. 1 A. Suzuki et al.
January 2002:91­98 p38 MAP kinase and osteoblastic cell differentiation
Erk can mediate activation of JNK induced by growth factors in
endothelial cells29
suggest that Erk could mediate the activation
of JNK and/or of p38 by FCS in MC3T3-E1 cells. However, our
results obtained with the specific MEK inhibitor appear to
exclude such a cross-talk between Erk and either JNK or p38 in
MC3T3-E1 cells. Indeed, the nearly complete inhibition of Erk
activation by 1 mol/L U0126 did not influence the stimulation
of either p38 or JNK by FCS (Figure 3A). Thus, the Erk pathway
does not seem to be involved in the activation of p38 and JNK
and the underlying molecular mechanism of activation of these
MAPKs in MC3T3-E1 cells by FCS remains to be investigated.
Analyses of the activation of the three types of MAPKs by
FCS during the various phases of proliferation and differentiation
of MC3T3-E1 cells indicate that the Erk pathway was stimulated
at all stages, whereas p38 and JNK were preferentially activated
during differentiation. The stimulation of p38 was observed in
the absence of any change in the amount of p38 expressed in
differentiating cells, suggesting that this response results mainly
from activation of upstream elements in this signal transduction
pathway. In contrast, the increase in JNK activity observed in
differentiating cells was associated with higher expression of
JNK2, suggesting that this MAPK may play a particular role in
osteoblastic cell differentiation.
To determine the role of Erk and p38 in regulating growth and
differentiation of MC3T3-E1 osteoblast-like cells, we used specific
inhibitors such as U0126 for the Erk pathway and
SB203580 to inhibit p38 activity. As mentioned earlier, Erk is
activated by growth factors acting through different types of
receptors and mediates their mitogenic effects in bone-forming
cells. As expected from this information, inhibiting the stimulation
of Erk in response to FCS in late-proliferating MC3T3-E1
cells stopped their replication (see Results), thus confirming the
importance of this pathway in controlling bone cell proliferation.
A role of Erk in osteoblastic cell differentiation has also been
proposed. Essentially, Takeuchi and coworkers suggested that, in
MC3T3-E1 cells, the binding of type I collagen to 2 1 integrin
receptors activates Erk, and that this signaling pathway mediates
extracellular matrix-dependent stimulation of ALP.37
More recently,
Xiao et al. reported that, in MC3T3-E1 cells, the Erk
pathway is essential for the regulation of Cbfa1, a transcription
factor involved in osteoblast differentiation, inferring from these
results that the Erk pathway may play an important role in the
establishment of the osteoblastic phenotype.40
Our data indicate
that the selective inhibition of Erk activity in early-differentiating
MC3T3-E1 cells had no major effect on expression of the main
markers of osteoblastic cell differentiation, namely ALP, Coll I,
and OC. If anything, it slightly enhanced the expression of ALP
and Coll I and induced a small and transient reduction in the
production of osteocalcin in early differentiating cells (Figure
3D). From this observation, we conclude that Erk is not essential
for expression of the three well-characterized differentiation
markers in MC3T3-E1 cells. However, because Erk is activated
by serum factors throughout the differentiation of MC3T3-E1
cells, this MAPK pathway is likely to play some yet unknown
role(s) during osteoblast maturation. For instance, it has been
described recently that Erk is involved in the synthesis of
interleukin-6 induced by either prostaglandin F2
38
or endothe-
lin-1,13
suggesting that Erk could play a role in mediating
osteoblast-osteoclast interaction.
Recent information from our laboratory has suggested that the
p38 pathway is involved in the differentiation of osteoblastic
cells. Indeed, we observed that p38 mediates the stimulation of
ALP activity induced by an agonist of Gi-protein-coupled receptors
in MC3T3-E1 cells.34,35
Results from the present study
suggest that p38 controls the expression of ALP in differentiating
MC3T3-E1 cells, as indicated by the good correlation between
the inhibition of ALP expression and that of p38 activity by
various doses of SB203580. Whether this signaling pathway
plays any significant role in controlling the expression of ALP in
vivo remains to be documented. In MC3T3-E1 cells, inhibition
of p38 activity was associated with a slight increase in their
proliferation, indicating that the p38 pathway is probably not
involved in mediating the growth arrest that precedes the onset of
preosteoblastic cell differentiation. These results further suggest
that the actions of the Erk and p38 pathways do not overlap.
Whether p38 also influences the expression of osteoblastic differentiation
markers other than ALP remains unclear. At high
doses (10­30 mol/L) of SB203580, the p38 activity was almost
completely inhibited and this effect was associated with a significant
reduction in Coll I and OC expression. However, because
these doses of SB203580 also inhibited JNK activity,
confirming a previous report that this inhibitor can also affect
Figure 6. Dose-dependent effect of SB203580 on collagen deposition,
osteocalcin production, and JNK activity in MC3T3-E1 cells. (A)
MC3T3-E1 cells were cultured for 4 days before exposure to various
concentrations of SB203580 (hatched bars, dashed bars) or its vehicle
(open bars) for 7 days and the amount of deposited collagen (hatched
bars) and the production of osteocalcin (dashed bars) were determined as
described in Materials and Methods. Values are the mean SEM of four
determinations of a representative experiment. *p 0.01 compared with
control. (B) MC3T3-E1 cells cultured for 10 days were preincubated with
various concentrations of SB203580 for 1 h. Then fresh MEM 10%
FCS culture medium containing either SB203580 or its vehicle were
added for a further 60 min and cells were rapidly frozen in liquid nitrogen
before lysis at 4°C. Phosphorylated c-Jun (p-c-Jun) in cell lysates was
determined by western blotting using a specific antibody. Changes in
c-Jun phosphorylation were quantified by densitometry (OD).
96 A. Suzuki et al. Bone Vol. 30, No. 1
p38 MAP kinase and osteoblastic cell differentiation January 2002:91­98
some particular JNKs,19
it is difficult to assess which of these
two MAPKs regulates Coll I and OC expression during osteoblastic
cell differentiation. In favor of a role for JNK, we found
a relatively good correlation between inhibition of either Coll I
deposition or OC secretion and inhibition of JNK activity by high
doses of SB203580, suggesting that JNK is likely to also participate
in the control of osteoblastic cell differentiation. This
hypothesis, however, could not be investigated because of a lack
of reagent for this MAP kinase.
In conclusion, the results of the present study indicate that
MAPKs are essential mediators of MC3T3-E1 osteoblastic cell
differentiation induced by serum growth factors. The Erk pathway
is activated throughout the various development stages and
is critical for cell proliferation. In addition, Erk probably plays
other, presently undefined roles in osteoblastic cells. The p38
pathway is activated when cells start to differentiate and it
controls ALP expression. Whether this MAPK also mediates the
regulation of other osteoblastic markers remains to be further
investigated. Finally, the JNK pathway is activated by growth
factors throughout osteoblastic cell development and its activity
is highest during differentiation, but its functional role also
requires further investigation. Altogether, our observations suggest
that the stimulation of proliferation and differentiation of
osteoblastic cells by serum growth factors involves the coordinate
activation of several MAPKs that have specific and distinct
roles in the regulation of these cellular processes.
Acknowledgments: The authors are grateful to Pierre Apostolides and
Se´verine Cle´ment for their expert technical assistance. This study was
supported by the Swiss National Science Foundation (Grant
32-58937.99) and by a grant allocated by AETAS, the Foundation for
Research into Ageing, Geneva, with the financial support of the "Fondation
Silva-Casa, Bern."
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Date Received: June 21, 2001
Date Revised: August 6, 2001
Date Accepted: September 4, 2001
98 A. Suzuki et al. Bone Vol. 30, No. 1
p38 MAP kinase and osteoblastic cell differentiation January 2002:91­98