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Introduction
Genistein, a soybean phytoestrogen, has estrogen-like
activity in mammals, including anabolic effects on bone
through mechanisms that remain to be
elucidated[1]. Several studies have shown that the incidence of mammary and other
cancers is inversely related to the dietary intake of soy
containing genistein and other
phytoestrogens[2]. Unlike endogenous hormone
17β-estradiol (E2), genistein lacks
stimulation on mammary glands and uteri, which makes it a possible
alternative to estrogen replacement therapy for osteoporosis
after menopause. While public and scientific interest in
genistein and other phytoestrogens is growing, their
mechanism of action on bone cells has largely remained unclear.
Core binding factor 1 (Cbfa1), belonging to the runt
domain transcription factor family, is shown to be a master
regulator of osteoblastic differentiation. Komori
et al[3] observed that the gene-targeted disruption of
osteoblast-specific Cbfa1 in the in vivo mouse model blocked
skeleto-genesis, and that heterozygous mutations of Cbfa1 in both
humans and mice led to cleidocranial dysplasia, an
autosomal dominant disorder. As further evidence of its role in
differentiation, Ducy et al[4] showed that the forced
expression of Cbfa1 induced the expression of principal
osteoblast-specific genes, such as bone sialoprotein, osteopontin, and
osteocalcin in non-osteoblastic cells. Cbfa1 also plays an
important role in mouse bone marrow-derived mesenchymal
stem cells (BMSC), becoming the osteoblastic lineage
in vivo and in vitro.
p38 Mitogen-activated protein kinase (MAPK) is a
conserved subfamily of MAPK involved in inflammatory response,
stress response, cell growth and survival, as well as
differentiation of a variety of cell types. Several studies have suggested
that p38 MAPK is involved in osteoblastic
differentiation[5]. Our previous work has also shown that exposure of BMSC to
SB203580, the inhibitor of p38 MAPK, resulted in reduced
alkaline phosphatase (ALP) activity (a marker of early
osteoblastic differentiation) and calcium deposition (a marker of late
osteoblastic maturation)[6]. Because the p38 MAPK pathway
and Cbfa1 are essential for osteoblastic differentiation, we
inferred that the anabolic effect of genistein might be through
the p38 MAPK pathway and activates the osteoblastic-specific transcription factor, Cbfa1. In the present study, we
examined the role of p38 MAPK and Cbfa1 activity in
mediating the effects of genistein on osteoblastic differentiation by
using mouse BMSC cultures, a population of pluripotent cells
within the bone marrow microenvironment which has a
capacity to undergo osteoblastic differentiation and mineralization
in vitro.
Materials and methods
Chemicals Alpha minimum essential medium
(α-MEM), fetal bovine serum (FBS), trypsin-EDTA,
penicillin_streptomycin solution, and SDS were obtained from Gibco-BRL (Grand
Island, NY, USA). The phosphorylated p38 MAPK assay kit,
SB203580 was purchased from New England Biolabs (Beverly,
MA, USA). The TRIzol total RNA isolation kit and Prime-It II
random primer labeling kit were obtained from Stratagene (La
Jolla, CA, USA). Anti-NF-κB antibody and anti-Cbfa1
antibody were purchased from Santa Cruz Biotech,
Inc (Santa Cruz, CA, USA). Other regents, antibodies, and reagent kits
were purchased from Sigma Chemical Co (St Louise, MO, USA).
Cell culture The bone marrow cells were obtained from
8- to 10-week-old female mice of the Kunming strain
(Experimental Animal Center of Xiangya Medical College,
Central South University, Changsha, China), as previously
described[6]. Briefly, the femurs and tibias were dissected, the
ends of the bones were cut, and the marrow was flushed out
with 2 mL of ice-cold α-MEM containing 10% FBS
(v/v) by using a needle and syringe. A suspension of bone marrow
cells was obtained by repeated aspiration of the cell
preparation through a 22 gauge needle and then the cells were counted
with a hemocytometer. The cells were seeded onto 12-well
plates or 60 mm plates at a density of
1×107 cells/mL and cultured for 5 d in
α-MEM supplemented with 15% FBS (v/v), 100
U/mL sodium penicillin G, and 100 mg/L streptomycin sulfate
in a humidified incubator with 5% CO2 and 95% air at 37 °C.
On d 5, all non-adherent cells were removed with the first
medium change and then the adherent cells (representing
BMSC) were grown for additional periods of up to 12 d in the
differentiation medium (phenol red-free α-MEM containing
10% FBS [v/v] supplemented with 5 mmol/L
β-glycerophosphate and 25 mg/L ascorbic acid) for inducing the
osteoblastic differentiation of BMSC. In the subsequent experiments,
the beginning day of culture in the differentiation medium was
defined as d 0. The genistein (1 μmol/L) and genistein (1
μmol/L) concurrent with SB203580 (1 μmol/L) were
respectively added every 2 d and the medium was
replaced every 4 d thereafter.
ALP Cellular ALP activity was assayed according to our
previous study[7]. At the completion of the incubation period,
the cells were harvested after removing the medium, washed
twice with phosphate-buffered saline (PBS), and treated for
10 min with 0.25% trypsin to detach the cells. Enzyme
activity was determined colorimetrically after incubation at 37 °C
for 30 min with p-nitrophenylphosphate as the substrate at
pH 10.3; the optical density was read at 400 nm. The total
protein content was measured by the method of Bradford.
Quantitation of calcium deposition The calcification of
BMSC was assessed by a modification of the Wada
procedure[8]. The cultures were decalcified with 0.6 mol/L HCl for
24 h. The calcium content was determined by measuring the
concentrations of calcium in the HCl supernatant by the
o-cresolphthalein complexone method. After decalcification,
the cultures were washed with PBS and solubilized with 0.1
mol/L NaOH/0.1% SDS. Total protein content was measured
by the method of Bradford. The calcium content of the cell
layer was normalized to the protein content.
Electrophoretic mobility shift assay Nuclear extracts were
prepared from BMSC treated with vehicle alone or genistein in
the presence or absence of 1 μmol/L SB203580. Synthetic
double-strand oligonucleotides of consensus Cbfa1 binding sequence
5'-AGCTGCAATCACCAACCACAGCA-3' was end-labeled with
(g_32P)ATP using the T4 polynucleotide kinase. Nuclear
extracts were incubated with binding buffer containing 20 mmol/L
Tris-HCl (pH 8), 10 mmol/L NaCl, 3 mmol/L EDTA, 0.05% Nonidet
P-40, 5 mmol/L dithiothreitol, 5% glycerol, and 1 mg poly (dI-dC)
for 15 min, after which the labeled probe was added and
incubated for an additional 15_20 min. The samples were subjected
to electrophoresis at room temperature on chilled 5%
polyacrylamide gel. For experiments using the anti-Cbfa1 antibody, the
antibody was added to the reaction mixture for 30 min prior to the
addition of the labeled probe. The samples were subjected to
electrophoresis at room temperature on 5% polyacrylamide gel,
which was dried and visualized by autoradiography.
RT-PCR Total RNA was extracted from cells using the
TRIzol total RNA isolation kit (Stratagene, La Jolla, CA, USA).
The RNA isolated from 8 d cultured cells was used for the
analyses of ALP and bone sialoprotein (BSP) expression,
whereas RNA isolated from 12 d cultured cells was used for
the analyses of osteocalcin (OC) expression. The RNA (3
μg) was reverse-transcribed in 50 μL reaction buffer
containing 0.5 mmol/L each of dNTP, 50_80 units of RNase block, 50
units of Moloney murine leukemia virus reverse
transcri-ptase, and 750 ng oligo(dT) for 90 min at 37 °C.
PCR using primers specific for ALP and BSP (the early
makers of osteoblastic differentiation), OC (the late osteogenic
marker), and GAPDH (internal control) were carried out in a
volume of 10 μL with 1×Pfu polymerase buffer, 190
μmol/L dNTP, 28 ng of each primer, 0.45 units Pfu polymerase, 0.18
μCi (α-32P)dCTP, and 1.0 μL of the template (from the above 50
μL RT reaction). Thermal cycling was carried out for 21 cycles
(GAPDH) or 30 cycles (ALP, BSP, and OC) at 62 °C, annealing
for ALP, BSP, and GAPDH, and 72 °C annealing for OC. The
amplified fragments were isolated on polyacrylamide gel (29:1
acrylamide to bis-acrylamide) and the autoradiographs were
scanned with the Gel Doc 2000 system (Bio-Rad, Hercules,
CA, USA). The primers used for the PCR amplification were
designed by computer assistance according to GeneBank:
ALP: forward, 5'-CTTGCTGGTGGAAGGAGGCAGG-3' and reverse, 5'-CACGTCTTCTCCACCGTGGGTC-3'; BSP:
forward, 5'-CTCGGGTGTAACAGCTAGCTAC-3' and reverse,
5'-CGTTCAGAAGGACAGCTGTCTG-3'; OC: forward, 5'-CT-CTGTCTCTCTGACCTCACAG-3' and reverse,
5'-GGAGCT-GCTGTGACATCCATAC-3'; and GAPDH: forward,
5'-TGGC-ACAGTCAAGGCCTGAGA-3' and reverse,
5'-CTTCTGA-GTGGCAGTGATGG-3'.
Northern blot analysis The BMSC grown in the 60 mm
dishes were treated at subconfluence with vehicle alone or
genistein in the presence or absence of 1 μmol/L SB203580
for the indicated period, and total RNA was isolated using
TRIzol total RNA isolation kit. The RNA isolated from the 8
d cultured cells were used for the analyses of osteopontin
(OPN) expression, whereas the RNA isolated from the 4 d
cultured cells were used for the analyses of Cbfa1 expression.
The total RNA (10 μg) samples were separated on 1%
agarose gels and transferred overnight to nitrocellulose
mem-branes, which were cross-linked with UV light. The
membranes were hybridized overnight at 58 °C with the
32P-labeled probe prepared according to the protocols of the
Prime-It II random primer labeling kit (Stratagene). The membranes
were washed twice at room temperature for 20 min with 2×
SSC (1×SSC=150 mmol/L NaCl, 15 mmol/L Na citrate, pH 7.0)
with 0.2% SDS and twice at 58 °C for 20 min with 1×SSC with
0.2% SDS before autoradiography. After stripping with
0.1×SSC with 0.1% SDS, the same membranes were probed
with mouse 28S rRNA using 55 °C hybridization overnight
and washed with 2×SSC with 0.1% SDS for 5 min at room
temperature once and 0.5×SSC with 0.1% SDS for 30 min twice
at 55 °C.
Western blot analysis Western blotting was carried out by
using the phosphorylated p38 MAPK assay kit according to
the manufacture's protocol. Briefly, the cells were lysated in
SDS sample buffer (62.5 mmol/L Tris-HCl, 2% SDS, 10% glycerol,
and 50 mmol/L dithiothreitol). After being sonicated and
denatured by boiling for 5 min, the protein concentration was
measured by the method of Bradford. 25 μg of proteins were
separated on 10% SDS_PAGE and transferred to a polyvinylidene
difluoride membrane (Millipore Corp, Bedford, MA, USA).
Non-specific binding sites on the membrane were blocked for 1 h in
blocking buffer (50 mmol/L Tris-HCl, 0.1% Tween 20, and 5%
non-fat milk). The membrane was exposed to a primary
monoclonal antibody of phosphorylated p38 MAPK or
non-phosphorylated p38 MAPK (1:1000 dilution) overnight at 4 °C and
then exposed to the second antibody (horseradish
peroxidase-conjugated, 1:2000 dilution) for 1 h. Detection was performed
with an enhanced chemiluminescence (ECL) reagent.
For the detection of the expression level of Cbfa1, nuclear
extracts (15 μg) prepared from cells at d 8 were separated on
10% SDS_PAGE and electrotransferred to a PVDF membrane.
The blots were probed with an anti-Cbfa1 antibody at a
dilution of 1:500 for 2 h at room temperature. The Cbfa1 protein
was detected by ECL according to manufacturer's
recommendations (Amersham Pharmacia Biotech, Tokyo, Japan).
Statistical analysis The data were the mean values of at
least 3 different experiments and expressed as mean±SD.
Student's t-test was used to compare data.
P<0.05 was considered statistically significant.
Results
Effects of genistein on p38 MAPK activation
The exposure of BMSC to genistein (1 μmol/L) resulted in the rapid
and sustained activation of p38 MAPK, which reached its
maximal activation at 5 min and even lasted for 360 min after
exposure (Figure 1A). The genistein-induced activation of
p38 MAPK was in a dose-dependent manner and 1 μmol/L
genistein was found to give the maximal response (Figure
1B). The activation of p38 MAPK was significantly
attenuated when genistein was incubated with 1 μmol/L of p38
MAPK inhibitor SB203580 (Figure 1C).
Effects of genistein on osteoblastic differentiation and
mineralization The BMSC were continuously cultured with
vehicle or genistein (1 μmol/L), and ALP activity and
calcium deposition were measured over a period of 0, 4, 8, and
12 d. Genistein markedly increased ALP activity and the
calcium deposition of the BMSC culture as a function of
cultured time compared with the control (Figure 2). The
increased ALP activity and calcium deposition could be
abolished by concurrent treatment with SB203580 (Figure 2).
There were slight, but significant reductions on ALP activity
and calcium deposition when SB203580 was used alone.
Effects of genistein on Cbfa1 DNA binding activity
Cbfa1-consensus DNA binding activity was markedly increased in
the genistein-treated cells compared with the untreated
control. However, the DNA binding activity was diminished
in the presence of the p38 MAPK inhibitor (Figure 3A). The
addition of the anti-Cbfa1 antibody resulted in an upper
super-shifted band and a decrease in the lower binding complex,
indicating that the binding was apparently specific to Cbfa1,
since an irrelevant antibody(anti-NF-κB antibody) had no
effect (Figure 3B).
Effects of genistein on the transcription of
osteoblastic differentiation genes The RT-PCR analysis showed that
the expression of ALP and BSP (the earlier makers of
osteoblastic differentiation at d 8) and OC (the later osteogenic
marker at d 12) were upregulated in genistein-treated BMSC
cultures. In contrast, SB203580 clearly inhibited the
genistein-mediated osteoblastic gene expression (Figure 4).
Effects of genistein on Cbfa1 expression
The Western blot analysis showed that the protein level of Cbfa1,
detected as 2 immunoreactive species of 60 and 65 kDa sizes,
was significantly increased in the genistein-treated samples
compared with the control. The increased Cbfa1 protein
level was also reduced by SB203580 treatment (Figure 5A).
The Northern blot analysis showed that the mRNA levels of
Cbfa1 at d 4 and OPN at d 8 were markedly upregulated in the
genistein-treated cells. Again, SB203580 treatment clearly
inhibited the genistein-mediated osteoblastic Cbfa1 and its
downstream gene OPN gene expressions (Figure 5B).
Discussion
In the present study, we utilized mouse BMSC to test the
hypothesis that genistein can stimulate osteoblastic
differentiation through the p38 MAPK_Cbfa1 pathway. The results
of our experiments demonstrated that: (i) the incubation of
mouse BMSC with genistein resulted in enhanced
osteoblastic differentiation as evidenced by increased ALP activity and
calcium deposition. At the same time, genistein treatment led
to the activation of the p38 MAPK pathway and increased
Cbfa1 message as well as the DNA binding ability; (ii) the
increased Cbfa1 DNA binding correlated with the activation
of several downstream Cbfa1-regulated genes, including ALP,
BSP, OPN, and OC; and (iii) the genistein-mediated increase
of Cbfa1 DNA binding enhanced osteoblastic differentiation,
and induced Cbfa1-regulated gene expressions were
remarkably attenuated by SB203580 treatment. These data provide
the first evidence that genistein is capable of increasing Cbfa1
activity through the p38 MAPK pathway, which is
responsible for its induction of osteoblastic differentiation in BMSC
cultures. This finding has important ramifications for the
therapeutic potential of genistein as an anti-osteoporosis agent.
Several studies have focused attention on the effect of
genistein on the MAPK pathway and reached different
conclusions. Kansra et al[9] found that the exposure of
intact platelets to genistein resulted in the activation of the
p44/42 MAPK pathway, which was due to the inhibition of
basal tyrosine kinase. Frey et
al[10] demonstrated that the p38 MAPK pathway was involved in genistein-inhibited cell
proliferation. In addition, some previous studies have shown
that at low concentrations (<=1 μmol/L), genistein has ER
(estrogen receptor)-dependent effects on osteogenesis, the
effects are similar to those of E2. At a higher concentration
(>10 μmol/L), however, genistein has anti-estrogenic actions,
namely, it downregulates osteogenesis, which is opposite
to the E2-induced
effects[11]. In our current study, genistein
showed a dose-dependent effect (0.01_1 μmol/L) on the
activation of p38 MAPK, and 1 μmol/L genistein was found to
give maximal stimulation, so genistein at a concentration of 1
μmol/L was used for further investigation in this study. The
specific inhibitor of the p38 MAPK kinase, SB203580, which
has little effect on other kinases, including the other
members of the MAPK family, was used to block the activation
of p38 MAPK. Our data show that SB203580 was able to
block genistein-mediated osteoblastic differen-tiation,
suggesting that p38 MAPK activation is critical for
genistein-induced osteoblastic maturation. We also found that
genistein does not profoundly affect ERK (extracellular
signal-regulated kinase) activity, another important member of
the MAPK pathway. PD98059, the specific inhibitor of the
ERK kinase had no effect on osteoblastic differentiation in
mouse BMSC cultures, suggesting that the ERK pathway is
unlikely to be involved in genistein-induced osteoblastic
differentiation (data not shown). In fact, our previous work
has demonstrated that ERK is important in cell proliferation
and genistein has little or no effect on the ERK
pathway[6].
Since Cbfa1 is a master regulator of osteoblastic
differen-tiation, we examined the effect of genistein on Cbfa1 activity.
Our results showed that Cbfa1 was activated by genistein at
both the mRNA and protein levels, which are associated with
observed increased DNA binding activity. We noticed that the
regulation of mRNA occurred at the earlier stage (d 4) of the
culture period, while the protein level of Cbfa1 did not change
until the later stage (d 8) of the culture period. One possible
explanation for this is that the increased protein level is a result
of the increased mRNA level induced by genistein in the earlier
stage. Similar regulation of Cbfa1 at the mRNA level has also
been reported with
1,25(OH)2-D3 and BMP(bone
morphogenetic protein) treatment in osteoblastic
cells[4].
In order to examine the connection between the increased
Cbfa1 activity and the activation of the p38 MAPK pathway
in genistein-treated cells, we used SB203580 to block the
activation of the p38 MAPK pathway and then check the
Cbfa1 activity. Our results showed that the increased
expression of Cbfa1 (both at the transcription and expression
levels) by genistein was significantly attenuated by SB203580
treatment. Moreover, the inhibitory effect of SB203580 also
affected the expression of several downstream
Cbfa1-regulated genes induced by genistein, including ALP, BSP, OPN,
and OC suggesting that p38 MAPK activation is upstream
of Cbfa1 activity. Similar results have been reported by
Jadlowice et al[12], which showed that p38 MAPK is involved
in the gene expression levels of Cbfa1 and osteoblastic
differentiation of human adult mesenchymal stem cells by
phosphophoryn, a non-collagenous dentin ECM
(extracel-lular matrix) protein and SIBLING (small integrin-binding
ligand glycoprotein) protein family member. The p38 MAPK
pathway is a major point of convergence for a variety of
intracellular signals initiated by the growth/differentiation
factor binding to receptor tyrosine kinases and mechanical
stimulation. In addition, there is a considerable cross-talk
between this pathway and other signaling events, such as
those mediated by protein kinase C[13] and
BMP[14].
Finally, our data indicate that genistein stimulates
osteoblastic differentiation through the p38 MAPK_Cbfa1 pathway.
These results provide new insight into the mechanistic basis
of osteoblastic differentiation by drugs such as genistein.
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