vitisin b stimulates osteoblastogenesis via estrogen ... · 84 vitisin b stimulates...
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J Chin Med 30(1): 82-98, 2019DOI: 10.3966/241139642019063001005
Original Article
Vitisin B stimulates osteoblastogenesis via estrogen receptor-mediated activation of non-genomic Src and
MAPKs pathway
Yu-Ling Huang 1, 2, Ling-Chiu Huang 1, Wen-Fei Chiou 1, 3, * 1 National Research Institute of Chinese Medicine, Taipei, Taiwan
2 Department of Cosmetic Science, Chang Gung University of Science and Technology, Taoyuan, Taiwan3 Ph.D Program in Clinical Drug Development of Chinese Herbal Medicine, Taipei Medical University, Taipei, Taiwan
Background/Purpose: Vitisin B is a major component existed in Vitis thunbergii, a herbal medicine used in Taiwan for treatment of inflammatory bone diseases. We recently reported that vitisin B stimulated differentiation in primary cultured osteoblasts and treatment with vitisin B-enriched preparation obviously ameliorated ovariectomy-induced bone loss in mice. This study further delineated the action mechanism(s) that how vitisin B stimulates osteoblastogenesis by using MC3T3-E1 osteoblasts. Methods: Cell differentiation and mineralization were identified by alkaline phosphatase (ALP) activity and Alizarin red S staining, respectively. RT-PCR and western blot were used to analyze the expression of osteoblast-associated genes and signal molecules. The transcriptional activity of estrogen receptor (ER) was also assessed. Results: Vitisin B significantly increased ALP activity, bone mineralization, mRNA expression of osteoids (type 1 collagen, bone sialprotein and osteocalcin) and bone-characteristic transcription factors (runt-related transcription factor-2 and osterix) through ER since such responsiveness were obviously repressed by ER antagonist ICI182,780. Unlike 17β-estradiol (E2), vitisin B failed to stimulate either ERα- or ERβ-mediated transcriptional activity. Nevertheless, vitisin B rapidly induced ERα and Src phosphorylations within 5 min and evoked late phosphorylations of p38 and ERK after 15-30 min stimulation through ER. Furthermore, p38 inhibitor SB203580 and MEK inhibitor PD98059 significantly inhibited vitisin B induced differentiation. Src inhibitor PP2 significantly repressed vitisin B-induced activation of MAPK and final mineralization suggesting that Src might play an important role. Conclusions: Vitisin B
*�Correspondence author: Wen-Fei Chiou, National Research Institute of Chinese Medicine, No.155-1, Section 2, Li-Nung Street, Peitou, Taipei 112, Taiwan, ROC. Tel: +886 2 28201999 ext.4481; Fax: +886 2 28250743; Email: [email protected]
Received 13th July 2018, accepted 3rd September 2018
83Yu-Ling Huang, Ling-Chiu Huang, Wen-Fei Chiou
might act through ER-mediated activation of Src and downstream MAPK to stimulate osteoblastogenesis which contributed to its beneficial effect in prevent bone loss.
Key words: estrogen receptor; osteoblast; Src kinase; vitisin B
Introduction
Osteoporosis is a reduction in bone mass
due to an imbalance between bone resportion
and bone formation. It can be treated by either
anti-osteoporotic drugs (anti-resorptives),
anabolic drugs (bone formation), or both [1,2].
Many plant-derived compounds have the
potential to counteract the deleterious effects of
estrogen deficiency on bone. Members of this
class are the so-called phytoestrogens. Estrogen
receptors (ERs) stimulate classical effects by
acting as nuclear transcription factors as well
as non-classical effects by activating distinct
cytoplasmic protein kinase cascades. Multiple
lines of evidence suggest that activation of
the tyrosine kinase c-Src represents one of the
initial steps in ER-α-mediated cell signaling
[3]. Numerous studies have demonstrated that
estrogens induce rapid and transient activation
of the c-Src phosphorylation cascade. Activation
of this cascade triggers vital cellular functions
including cell proliferation and differentiation.
The essential role of c-Src in the non-classical
action of steroid receptors was demonstrated in
experiments with embryonic fibroblasts derived
from c-Src-/- mice [4].
Vitisin B is a dominant stilbene contained
in the root of Vitis thunbergii Sieb. & Zucc.
(Vitis ficifolia Bge. Vitaceae), a well-known
herbal remedy traditionally used in Taiwan for
diarrhea, jaundice, hepatitis, fracture and injury
[5]. Accumulating use experience suggests an
alcoholic drench of the roots of V. thunbergii
also helps cure bone fractures, contusions and
prevents bone loss, suggesting that this herb
might either stimulate new bone formation or
inhibit bone resorption. We recently reported that
orally administered with (+)-vitisin B-enriched
preparation (VtR) significantly ameliorated
the deterioration of bone mineral density in
ovariectomized mice and found that vitisin B
could stimulate osteoblast function in cultured
bone marrow cells [6]. Considering that vitisin
B might display protective effect against bone
loss, this study aimed to clarify how vitisin B
stimulates osteoblastogenesis.
Materials and methods
1. Chemicals and antibodiesFetal bovine serum (FBS), charcoal-
stripped FBS (sFBS), α-modified minimum
essential medium (α-MEM), TRIzol reagent,
penicillin and streptomycin were purchased
from Invitrogen (Carlsbad, CA). 17β-Estradiol
(E2), ICI 182,780 (Fulvestrant, (7a,17b)-7-[9-
[(4,4,5,5,5pentafluoropentyl)Sulfinyl]nonyl]
estra-1,3,5(10)-triene-3,17-diol), ascorbic
ac id , 3 - (4 ,5-d imethyl - th iazo l -2y l ) -2 ,5-
84 Vitisin B stimulates osteoblastogenesis
diphenyltetrazolium bromide (MTT), Alizarin
Red S and p-nitrophenyl phosphate (p-NPP)
were purchased from Sigma Chemical Co. (St.
Louis, MO, USA). PP2 (AG 1879, 4-Amino-
5-(4-chlorophenyl)-7-(t-butyl)pyrazolo [3,4-
d]pyrimidine) was purchased from Merck
(Darmstadt, Germany). β-glycerophosphate
was obtained from Wako Pure Chemicals
(Osaka, Japan). Antibodies against total and
phosphorylated c-Src were purchased from Cell
Signaling Technology (Beverly, MA, USA). The
other antibodies were obtained from Santa Cruz
Biotechnology (Santa Cruz, CA). Enhanced
chemiluminescence (ECL) Western blotting
detection reagent was purchased from GE
Healthcare (Little Chalfont, Buckinghamshire,
UK).
2. Extraction and isolation of vitisin BVitis thunbergii was purchased in Taipei,
Taiwan, in July 1998 and identified by Mr.
Jun-Chih Ou, a taxonomist retiring from our
institute. A voucher specimen (NRICM-98-010)
is deposited at the Herbarium of National
Research Institute of Chinese Medicine. Detailed
information for the HPLC analysis and the
structure identification of (+)-vitisin B (purity
99.4%) was described in our previous work [7,
8].
3. Cell culture, differentiation and miner-alization
Murine osteoblast ic MC3T3-E1 cell
exhibited high levels of known osteoblast
markers. This provides a suitable model to
analyze osteoblast function and related signaling
pathway since osteoblastogenic process can
be easily and quickly assessed. Cells were
maintained in α-MEM as described previously
[9]. For differentiation or mineralization, cells
seeded in 96- or 24-well plates were cultured
to reach confluence. After that, the medium
was replaced to phenol red-free MEMα
supplemented with 1% sFBS, 50 μg/mL ascorbic
acid and 10 mMβ-glycerophosphate (define
as differentiation medium) for 24 h. After
that, cells were stimulated with vitisin B, E2
or vehicle for different time periods to induce
differentiation (3-8 days) or mineralization (7-28
days). Osteoblastic differentiation was assessed
by measuring the alkaline phosphatase (ALP)
activity and represented either as μM p-NPP/
mg protein/hour or indicated as % of control
(maximum response is defined as 100%) [10].
The calcified bone nodules (mineralization)
appeared bright red in color by Alizarin red S
staining was observed under light microscope
and photographed. Following, calcium precipitate
was extracted by 0.02N sodium hydroxide
and the dissolved product was determined by
measuring the absorbance at 548 nm [10].
4. RNA preparation and reverse transcri-ptase polymerase chain reaction (RT-PCR)
The total cellular RNA was isolated using
TRIzol reagent and RT-PCR was performed
using RevertAidTM First Strand cDNA Synthesis
Kit and Go Taq® Flexi DNA Polymerase
according to the manufacturer’s instruction.
Mouse-specific primer sequences for sense or
antisense strand of osteocalcin (OCN), bone
sialprotein (BSP), type 1 collagen (Col-1), runt-
85Yu-Ling Huang, Ling-Chiu Huang, Wen-Fei Chiou
related transcription factor-2 (Runx2), osterix
and GAPDH were described in our previous
study [11, 12].
5. Transient transfection and ER-mediated luciferase activity assay
T h e c e l l s w e r e t r a n s f e c t e d b y
LipofectamineTM 2000 reagent. ER-α, ER-β and
estrogen response element (ERE)-containing
luciferase reporter plasmid vERETkluc were
purchased from Clontech (Mountain View, CA,
USA). 0.4 μg ER-α or ER-β plasmid, 0.4μg
vERETkluc, together with 0.1 μg internal control
reporter plasmid pRL-TK, a Renilla luciferase
control vector, was cotransfected into the cells
in triplicate. Five hours after transfection, cells
were treated with vehicle, E2 or vitisin B for 24
h. After treatment, the cells were lysed and the
luciferase activity was measured using the Dual
Luciferase Reporter assay System and the signal
was detected by TD-20/20 Luminometer (Turner
Design, Sunnyvale, CA, USA). The estrogen
promoter activity was expressed as firefly
luciferase values normalized to pRL-TK Renilla
luciferase values.
6. ImmunoblottingTreated cells were harvested and lysed with
lysis buffer [11, 12]. Equal amounts of proteins
were separated by SDS-PAGE and transblotted
onto PVDF membranes (Immobilin-P, Millipore
Corp., Danvers, MA, USA). After blocking the
non-specific binding sites, the blots was probed
with first antibodies and followed by incubation
with secondary antibodies. The antigen-antibody
complexes were then detected with ECL reagent.
7. Statistical analysis
The results are expressed as mean ± Sp.E.
Statistical analysis was performed with an
analysis of variance followed by the one-way
analysis of variance (ANOVA) followed by
Newman-Keuls test for multiple comparisons.
A value of p<0.05 is accepted as statistically
significant difference.
Results
1. Effects of vitisin B on osteoblastic differ-entiation and mineralization
This study aimed to clarify whether vitisin
B stimulated MC3T3-E1 differentiation as
did in primary cultured osteoblasts. Vitisin
B significantly stimulated ALP activity in
MC3T3-E1 cells in time-dependent (Fig. 1A)
and concentration-dependent (Fig. 1B) manner
but without detectable effect on cell proliferation
or viability (data not shown). About 2-folds and
2.2-folds induction was reached after cultured
with 10-20 μM vitisin B and 100 nM E2 for 8
days, respectively. Because vitisin B-evoked
osteogenic effects in MC3T3-E1 cells were equal
potent as did in primary cultures [6], MC3T3-E1
cell was thus used for mineralization assay and
the others.
As shown in Figs. 2A & 2B, addition
of vitisin B (0.5-20μM) time-dependently
and concentration-dependently enhanced
mineralization when compared with control
(differentiation medium alone). Significant
enhancement in mineralization was occurred up
to 1 μM of vitisin B and a plateau effect (~4-
fold) noted at 10-20 μM. 100 nM of E2 also
86 Vitisin B stimulates osteoblastogenesis
(A) Vitisin B stimulated the increase in ALP activity in time- and concentration-dependent manner. (B) Osteogenic effect of vitisin B was compared with 17β-estradiol (E2, 100 nM). Each value is the mean ± SE
of six independent experiments. * p<0.05, ** p<0.01 and *** p<0.001, compared with cell incubated with differentiation medium alone.
Figure 1 Effect of vitisin B on ALP activity in MC3T3-E1 cells.
(A) (B)
(A) Vitisin B stimulated the increase in bone mineralization in time- and concentration-dependent manner. (B) Effect of vitisin B on bone nodule formation was compared with 17β-estradiol (E2, 100 nM). The level of
mineralization was analyzed by AR-S staining as described in Material and Methods. Each value is the mean ± SE of six independent experiments. * p<0.05, ** p<0.01 and *** p<0.001, compared with cell incubated with differentiation medium alone.
Figure 2 Effect of vitisin B on bone mineralization in MC3T3-E1 cells.
(A) (B)
stimulated the formation of calcified nodules
by approximately 4-fold when compared to the
control. The mRNA expressions of osteoid (bone
matrix) (Fig. 3A) and osteoblast-characteristic
87Yu-Ling Huang, Ling-Chiu Huang, Wen-Fei Chiou
transcription factors (Fig. 3B) were also
concentration-dependently increased by vitisin B
(1, 5, 10 and 15μM) and E2 (1, 10 and 100 nM)
stimulation, respectively.
2. The roles of estrogen receptor (ER) in vitisin B-induced osteoblastogenesis
Estrogen receptor (ER) plays an important
role in regulation of osteoblastogenesis. The
result shown in Fig. 4A indicated that vitisin B
(10 μM) or E2 (100 nM) induced increase in
ALP activity were concentration-dependently
abolished by ICI-182,780. Furthermore, the
stimulatory effects of vitisin B on mineralization
Figure 3 Effects of vitisin B and 17β-estradiol (E2) on the mRNA expression of (A) osteoid (type 1 collagen, Col-1; bone sialprotein, BSP; osteocalcin, OCN) and (B) bone-characteristic transcription factors (runt-related transcription factor-2, Runx2 ; osterix, Osx) in MC3T3-E1 cells. mRNA levels were determined by RT-PCR.
(A)
(B)
(Fig. 4B) and mRNA expression of bone-
characteristic genes (Fig. 4C) were also
significantly reduced by ICI-182,780.
3. Effects of vitisin B on ER-mediated luciferase activities
100 nM of E2 significantly increased the
ERE-dependent luciferase activities via ERα
or ERβ (Fig. 5A). In contrast, vitisin B failed
to induce ERE-dependent luciferase activities
via ERs. Western blot analysis reconfirmed that
vitisin B did not induce ERα nor ERβ nuclear
translocation indicating that vitisin B-evoked
bone anabolic action was not via the activation
88 Vitisin B stimulates osteoblastogenesis
Figure4 Vitisin B-evoked increase in (A) ALP activity, (B) mineralization and (C) mRNA expression of bone-characteristic genes (Col-1: type 1 collagen; BSP, bone sialprotein; OCN, osteocalcin; Runx2, runt-related transcription factor-2; Osx, osterix) were significantly repressed by ICI-182,780 treatment. Each value is the mean ± SE of five independent experiments. * p<0.05, ** p<0.01 and *** p<0.001, compared with cell incubated with vitisin B alone or 17β-estradiol (E2) alone.
(B)
(A)
of ERE-dependent transcription (Fig. 5B).
4. Effects of vitisin B on ERα phosphor-ylation
Because vi t is in B fai led to act ivate
ERE-dependent transcription via ERs, we
hypothesized that it might alternatively activate
ER-mediated non-genomic signaling pathway to
induce osteoblastogenesis. Results showed that
89Yu-Ling Huang, Ling-Chiu Huang, Wen-Fei Chiou
Figure 5 Effects of vitisin B- and 17β-estradiol (E2) on ERs (α or β)-mediated transcriptional activities in MC3T3-E1 cells. (A) Transfected cells were treated with vehicle (control), E2 (100 nM) or vitisin B (5, 10 and 15 μM) as described in Material and Methods. The estrogen response element (ERE)-dependent luciferase activity was represented as the ratio versus control group (normalized to 1). Results were obtained from three independent experiments and expressed as mean ± SE. ** p<0.001 versus control. (B) MC3T3-E1 cells were treated with vitisin B (10 μM) for 5-30 min. Proteins extracted from cytosolic and nuclear lysates were analyzed by western blotting and probed with anti-ERs (α or β) antibodies.
(A)
(B)
cells stimulated with vitisin B (10 μM) or E2
(100 nM) for a short time rapidly increased the
expression ratio of phospho-ERα to ERα (pERα/
ERα) suggesting that vitisin B and E2 could
activate ERα by phosphorylation. As shown in
Fig. 6A (left), E2 (100 nM) increased pERα/
ERα within 5 min and remained up-regulated for
15 min treatment. Similarly, vitisin B (10 μM)
was able to significantly increase pERα/ERα at
1-5 min and the phosphorylation was sustained
90 Vitisin B stimulates osteoblastogenesis
throughout around 10-15 min of incubation. The
concentration-response for vitisin B and E2 on
ERα phosphorylation were shown in the right of
Fig. 6A.
5. ER-mediated early phosphorylation of c-Src is essential for vitisin B-induced
Figure 6 Vitisin B and 17β-estradiol (E2) both evoked time-related and concentration-related phosphorylations of ERα (A) and Src (B) in MC3T3-E1 cells and vitisin B-evoked such phosphorylations were significantly inhibited in the presence of ICI-182,780 (C). Proteins extracted from vitisin B- and E2-stimulated cells were analyzed by western blotting and probed with antibodies against ERα, phospho- ERα, Src, phospho-Src in the absence or presence of ICI-182,780, respectively.
(A)
(C)
(B)
91Yu-Ling Huang, Ling-Chiu Huang, Wen-Fei Chiou
osteoblastogenesisIncreasing evidence indicates that c-Src
might exert key functions in ERα mediated non-
classical effect of estrogens [3]. Whether E2 or
vitisin B act through c-Src pathway to induce
osteoblastogenesis was studied by evaluate the
phosphorylation (activation) of c-Src. Results
showed in Fig. 6B demonstrated that E2 (100
nM) and vitisin B (10μM) both increased tyrosine
phosphorylation of c-Src (Tyr 416) within 5
min stimulation. The concentration-response of
vitisin B and E2 on Src phosphorylation were
shown in the right of Fig. 6B. Interesting, vitisin
B induced c-Src phosphorylation was repressed
by ICI 182,780 treatment (Fig. 6C). This result
indicated that vitisin B-induced activation of
c-Src is ER-dependent. To further clarify whether
Src really participating in vitisin B-induced
osteoblastogenesis or not, a widely used, specific
inhibitor for all members of Src families PP2 was
applied. As shown in Fig 7, PP2 obviously and
concentration-dependently repressed not only the
Figure 7 Vitisin B-evoked increase in mRNA expression of bone-characteristic genes (BSP, bone sialprotein; Col-1: type 1 collagen; Runx2, runt-related transcription factor-2; Osx, osterix) and mineralization were significantly repressed by Src kinase inhibitor PP2. Each value is the mean ± SE of five to six independent experiments. * p<0.01 and ** p<0.001, compared with cell incubated with vitisin B alone.
92 Vitisin B stimulates osteoblastogenesis
expression of osteoblast-characteristic genes but
also the mineralization evoked by vitisin B.
6. Vitisin B-evoked activation of MAPKs is mediated by ER/Src and is necessary for vitisin B-induced osteoblas different-iation
Several studies indicated that MAPKs
were involved in regulation of osteoblastic
differentiation and maturation [13, 14]. We
also like to know whether vitisin B evoked
differentiation and maturation was mediated
by MAPKs or not. Results showed in Fig.
8A indicated that treatment with vitisin B
significantly increased the phosphorylation of
p38 and ERK, but not JNK (data not shown).
Overall, the activated pattern for MAPKs was
relative late than ERα or Src activation. As shown
in Fig. 8A, the time-dependent activation of p38
and ERK was obviously detected at15 min and
further up-regulated after 30 min stimulation and
such phosphorylations were obviously repressed
by ICI-182,780 and PP2 treatment (Fig. 8B),
respectively. These results suggested that ER and
Src are upstream components in the activation
of MAPKs by vitisin B. Subsequent functional
study was designed to further explore whether
Figure 8 Vitisin B activated p38 MAPK and ERK phosphorylations in time- and concentration-dependent manner (A) and such phosphorylations were significantly repressed by ICI-182,780 and PP2 (B). In (A), proteins extracted from vitisin B-stimulated cells were analyzed by western blotting and probed with antibodies against p38, phospho-p38, ERK, phospho-ERK and actin. In (B), transblotted membranes were reacted with antibodies against p38, phospho-p38, ERK, phospho-ERK, phosphor-Src and actin in the absence or presence of ICI-182,780 or PP2, respectively.
(A)
(B)
93Yu-Ling Huang, Ling-Chiu Huang, Wen-Fei Chiou
p38 MAPK or ERK signaling is necessary
for vitisin B-induced osteoblas differentiation
by using individual inhibitor (SB203580 and
PD98059). As shown in Fig. 9, vitisin B-evoked
increase in ALP activity was repressed by about
23%, 62%, 88% and 97% in the presence of 0.5,
1, 5 and 10 μM SB203580 and similar inhibitory
pattern was observed by PD98059 treatment.
Discussion
The present study clearly demonstrated
that vitisin B mimicked E2 in stimulating
osteoblast differentiation, expression of bone-
characteristic genes and induced mineralization
via ER in MC3T3-E1 cells, suggesting that
vitisin B could exert estrogen-like effects in
Figure 9 Vitisin B-stimulated ALP activity was significantly abolished by SB203580 and PD98059 treatment, respectively. Each value is the mean ± SE of four to five independent experiments. * p<0.05 and ** p<0.01, compared with cell incubated with vitisin B alone.
94 Vitisin B stimulates osteoblastogenesis
promoting osteoblastic functions. Evidence
indicates that estrogen-mediated responsiveness
is mediated by a complex interface of direct
control of gene expression (so-called “genomic
action”) and by regulation of cell signaling/
phosphorylation cascades (referred to as the
“non-genomic”, or “extranuclear” action) [15-
17]. ERs are the members of the superfamily of
ligand-regulated nuclear transcription factors.
ERα and ERβ have been identified in cultured rat
osteoblasts and estrogen was shown to stimulate
the differentiation of osteoblasts [18]. When
bound to E2, E2 stimulated ERα translocation
to the nucleus and activate the expression of
genes that have EREs in their promoter regions
[19]. Homozygous deletion of the ERα gene
in mice results in decreased longitudinal bone
growth, decreased cortical bone density, as
well as decreased bone formation, suggesting
that ERα is of critical importance in promoting
overall bone growth [20, 21]. Unlike E2, the
transfection study indicated that vitisin B failed
to induce ERE-dependent luciferase activities
via either ERα or ERβ. Results obtained from
western blotting also revealed that vitisin B
did not increase the nuclear translocation of
ERs. However, vitisin B could activate ERα
phosphorylation (but not ERβ) in MC3T3-E1
cells within 5 min of incubation as E2 did. ERα
can activate protein-protein signaling cascade
in the cytoplasm and to regulation of genes
expression in which instead of ERα entering the
nucleus to activate transcription [15]. Thus, we
hypothesized that vitisin B would act through
so-called “non-genomic action” to induce
osteoblastogenesis.
c-Src is known to play a pivotal role in
osteoblast differentiation, and c-Src null primary
osteoblasts appear to undergo ‘premature’
differentiation. Multiple lines of evidence suggest
that activation of the tyrosine kinase c-Src,
represents one of the initial steps in ER-mediated
cell-signaling [3]. In non-genomic pathway,
binding of E2 to membrane ER rapidly activates
c-Src kinase. Src undergoes an intermolecular
autophosphorylation at tyrosine 416, and its
phosphorylation promotes kinase activity
[22]. The present results indicated that vitisin
B induced phosphorylation of c-Src and such
phosphorylation was concentration-dependently
repressed by ICI-182,780 treatment. Recently,
c-Src was observed to serve as a transducer of
signaling events taking place in osteoblast cells
in response to resveratrol and diosgenin. [23,
24] Furthermore, the c-Src inhibitor dasatinib
decreases osteoblastic differentiation in human
bone marrow-derived mesenchymal stromal
cells, primary mouse osteoblasts and MC3T3-E1
cells [25, 26]. We also examine whether c-Src
signaling mediated the osteogenesis by vitisin
B in MC3T3-E1. Pharmacologic blockade of
c-Src activity by PP2 significantly blunted vitisin
B-induced expressions of bone-specific genes
and transcription factors and final mineralization.
The present results indicated that ER-mdiated
c-Src phosphorylation is required for vitisin
B-induced osteoblastic differentiation and
maturation in MC3T3-E1 cells.
Several studies demonstrated that Src/
MAPKs influenced osteoblast differentiation and
95Yu-Ling Huang, Ling-Chiu Huang, Wen-Fei Chiou
maturation. The essential role of Src kinase in
the non-genomic action of steroid receptors was
demonstrated in experiments with embryonic
fibroblasts derived from Src−/− mice. These cells
did not show rapid activation of the MAP kinase
pathway in response to androgen receptor and
ERα activation, whereas wild-type Src+/+ cells did
[4]. Suzuki et al. (1999) showed that ERKs play
an important role in cell replication, whereas
p38 MAPK participates in the regulation of
ALP expression during osteoblast differentiation
[27]. Liao et al. found that genistein induced
the expression of ALP, BSP and OCN through
activation of p38 MAPK pathway [28]. Tang
et al. reported that imperatorin and bergapten
enhance ALP activity, type I collagen synthesis
and mineralization in rat via the p38- and ERK-
dependent signaling pathway [29]. The present
results showed that treated the cells with vitisin
B induced the phosphorylation of p38 MPAK
and ERK. Furthermore, inhibition of p38 MAPK
and ERK activation by either SB203580 or
PD98059 effectively decreased vitisin B-induced
ALP activity. Collectively, the present findings
suggested that activation of p38 MAPK and ERK
were involved in vitisin B regulated osteoblastic
differentiation.
We recently reported that orally admin-
istered with VtR significantly ameliorated
the deterioration of bone mineral density in
ovariectomized mice [6]. The present study
further clarified the (+)-vitisin B could stimulate
osteoblastogenesis via ER-mediated activation
of non-genomic Src and MAPKs pathway. In
conclusion, our findings provide the evidence
to support the use of VtR as a potential botanic
remedy for osteoporosis therapy.
Funding/support statement
This work was supported by National
Science Council Foundation grant (NSC101-
2320-B-077-004-MY3 to W.F.C) and National
Research Inst i tute of Chinese Medicine
Foundation grant (NRICM100-DBCM-02 to
W.F.C and NRICM100-DMC-05 to Y.L.H).
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原始論文
Vitisin B 經由雌激素受體調節 Src 激酶及下游
MAPK 之非基因型訊息路徑而刺激骨質新生
黃鈺玲 1, 2、黃齡丘 1、邱文慧 1, 3, *
1衛生福利部國家中醫藥研究所,臺北,臺灣2長庚科技大學化妝品應用系,桃園,臺灣
3中草藥臨床藥物研發博士學位學程,臺北醫學大學,臺北,臺灣
Vitisin B 是小本山葡萄主要成分之一,民間常使用該本土植物於發炎性骨疾,
宣稱可強壯筋骨。我們先前發現餵食富含 vitisin B 的提取物可明顯改善卵巢切除
鼠的骨質流失現象,現在進一步利用 MC3T3-E1 造骨細胞,研究 vitisin B 對骨質
新生的影響。結果發現與骨分化 / 成熟有關的鹼性磷解酶活性及礦化程度,均因
處理 vitisin B 而明顯提高。vitisin B 也使骨基質以及特有轉錄因子的表現量增加,
且上述促進作用皆會被雌激素受體(ER)拮抗劑所抑制。不像典型的雌激素作用
劑 17-estradiol,vitisin B 不會刺激 ER 或 ER 調控的轉錄活性,但反而可快速磷酸
化 ER 受體及 Src 激酶、繼而磷酸化 p38 及 ERK 這二個 MAPK,這些作用也都會
被 ER 拮抗劑所抑制。進一步發現 p38 及 ERK 抑制劑會干擾 vitisin B 誘導的造骨
細胞分化,而 Src 抑制劑也會阻斷 vitisin B 誘導的 MAPK 活化與礦化。綜上,本
研究發現 vitisin B 具改善骨質流失的功效,其機轉可能是經由雌激素受體去活化
細胞內非基因路徑的 Src 激酶及下游的 MAPK 訊息分子,進一步啟動造骨細胞特
有的轉錄因子活化,繼而促進骨基質生成及礦化。
關鍵字:雌激素受體、造骨細胞、Src 激酶、vitisin B
* 通訊作者:邱文慧,衛生福利部國家中醫藥研究所,地址:台北市北投區立農街 2段 155-1號,電話:02-28201999分機 4481,傳真:02-28250743,Email: [email protected]
107 年 7 月 13 日受理,107 年 9 月 3 日接受刊載