vitisin b stimulates osteoblastogenesis via estrogen ... · 84 vitisin b stimulates...

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-


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


(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


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


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


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


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)


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.


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)


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.


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


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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J Chin Med 30(1): 82-98, 2019DOI: 10.3966/241139642019063001005

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

vitisin b stimulates osteoblastogenesis via estrogen ... · 84 vitisin b stimulates...

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