Caffeic acid 3,4-dihydroxy-phenethyl ester suppresses receptor activator of NF-κB ligand-induced osteoclastogenesis and prevents ovariectomy-induced bone loss through inhibition of

Caffeic Acid 3,4-Dihydroxy-Phenethyl Ester SuppressesReceptor Activator of NF-kB Ligand–InducedOsteoclastogenesis and Prevents Ovariectomy-InducedBone Loss Through Inhibition of Mitogen-ActivatedProtein Kinase/Activator Protein 1 and Ca2þ–NuclearFactor of Activated T-cells Cytoplasmic 1 SignalingPathways

Xian Wu,1* Zhenxi Li ,1,2* Zhengfeng Yang,1 Chunbing Zheng ,1 Ji Jing ,1 Yihua Chen ,1 Xiyun Ye ,1

Xiaoyuan Lian,1 Wenwei Qiu,3 Fan Yang,3 Jie Tang,3 Jianru Xiao,2* Mingyao Liu,1,2,4* and Jian Luo1,2*1Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences,East China Normal University, Shanghai, China

2East China Normal University and Shanghai Changzheng Hospital Joint Research Center for Orthopedic Oncology, Shanghai, China3Institute of Medicinal Chemistry and Department of Chemistry, East China Normal University, Shanghai, China4Alkek Institute of Biosciences and Technology, Texas A&M University Health Science Center, Houston, TX, USA

ABSTRACTReceptor activator of NF-kB ligand (RANKL) stimulation leads to the activation of mitogen-activated protein kinase (MAPK)/AP-1

and Ca2þ–nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) signaling pathways in osteoclastogenesis. Targeting these pathways

has been an encouraging strategy for bone-related diseases, such as postmenopausal osteoporosis. In this study, we examined the

effects of caffeic acid 3,4-dihydroxy-phenethyl ester (CADPE) on osteoclastogenesis. In mouse bone marrow monocytes (BMMs) and

RAW264.7 cells, CADPE suppressed RANKL-induced osteoclast differentiation and actin-ring formation in a dose-dependent manner

within non–growth inhibitory concentrations at the early stage, while CADPE had no effect on macrophage colony-stimulating factor

(M-CSF)-induced proliferation and differentiation. At the molecular level, CADPE inhibited RANKL-induced phosphorylation of MAPKs,

including extracellular signal-regulated kinases 1/2 (ERK1/2), p38, and c-Jun N-terminal kinase (JNK), without significantly affecting the

NF-kB signaling pathway. CADPE abrogated RANKL-induced activator protein 1 (AP-1)/FBJ murine osteosarcoma viral oncogene

homolog (c-Fos) nuclear translocation and activation. Overexpression of c-Fos prevented the inhibition by CADPE of osteoclast

differentiation. Furthermore, CADPE suppressed RANKL-induced the tumor necrosis factor receptor associated factor 6 (TRAF6)

interaction with c-src tyrosine kinase (c-Src), blocked RANKL-induced the phosphorylation of protein kinase B (AKT), and inhibited

RANKL-induced Ca2þ oscillation. As a result, CADPE decreased osteoclastogenesis-related marker gene expression, including NFATc1,

TRAP, cathepsin K, and c-Src. To test the effects of CADPE on osteoclast activity in vivo, we showed that CADPE prevented ovariectomy-

induced bone loss by inhibiting osteoclast activity. Together, our data demonstrate that CADPE suppresses osteoclastogenesis and bone

loss through inhibiting RANKL-induced MAPKs and Ca2þ-NFATc1 signaling pathways. CADPE is a novel agent in the treatment of

osteoclast-related diseases, such as osteoporosis. � 2012 American Society for Bone and Mineral Research.

KEY WORDS: CAFFEIC ACID; MAPK/AP-1; Ca2þ-NFATc1; MENOPAUSE; OSTEOPOROSIS

Introduction

Bone homeostasis is balanced between bone formation by

osteoblasts and bone resorption by osteoclasts, which

occurs throughout our lives.(1) Deficiency in bone formation by

osteoblasts or excessive bone resorption by osteoclasts can

cause a number of osteopenic and pathologic diseases,

especially osteoporosis.(2–4) Therefore, identification of agents

that either increase bone formation or block osteoclast

resorption are essential for the development of therapeutic

ORIGINAL ARTICLE JJBMR

Received in original form March 8, 2011; revised form January 30, 2012; accepted February 6, 2012. Published online February 15, 2012.

Address correspondence to: Jian Luo, PhD, East China Normal University, 500 Dongchuan Road, Shanghai 200241, China. E-mail: [email protected]

Additional Supporting Information may be found in the online version of this article.

*XW, ZL, and JX contributed equally to this work. ML and JL are co-senior authors.

Journal of Bone and Mineral Research, Vol. 27, No. 6, June 2012, pp 1298–1308

DOI: 10.1002/jbmr.1576

� 2012 American Society for Bone and Mineral Research

1298

drugs for osteoporosis. However, the drugs for the disease are far

from ideal.(5) For example, the therapeutic peptide parathyroid

hormone (PTH), which can induce bone formation, must be

given as an injection, and has a 2-year maximal treatment course

in clinic due to bone cancer concerns.(6) Bisphosphonates, which

can inhibit osteoclast resorption, however, are poorly absorbed

and can cause damage to the gastrointestinal tract.(7) In addition,

estrogen-replacement therapy, often prescribed for postmeno-

pausal women, has been proven to increase the risk for uterine

and breast cancer, heart attack, and blood clots, and its use

has been discouraged for long-term treatment.(8) Therefore,

there is considerable scientific and lay public interest in finding

alternative agents and treatments for osteoporosis.

Previous research in postmenopausal animals shows that the

main reason for postmenopausal osteoporosis is estrogen

deficiency accompanied by production of pro-osteoclastogenic

cytokines, such as tumor necrosis factor a (TNFa) and receptor

activator of NF-kB ligand (RANKL),(9) which strongly increases

osteoclast lifespan and activity.(4,10) Therefore, inhibition of

osteoclastogenesis is a promising strategy for postmenopausal

osteoporosis treatment. Osteoclasts are multinucleated cells

formed by the differentiation and fusion of bone marrow

monocytes (BMMs) and are the unique cells involved in bone

resorption.(11)

Previous studies indicated that two cytokines, macrophage

colony-stimulating factor (M-CSF) and RANKL are essential and

sufficient to promote osteoclastogenesis. M-CSF can induce the

proliferation of BMMs and their differentiation into osteoclast

precursors, and RANKL can subsequently induce the differentia-

tion of osteoclast precursors intomature osteoclasts.(2,12) Binding

of RANKL to its receptor RANK results in the recruitment of the

adaptor molecules TNF receptor-associated factors (TRAFs).(11)

The formation of the RANK-TRAFs complex mediates mitogen-

activated protein kinases (MAPKs) signaling pathways in

osteoclast formation.(13,14) In particular, RANK-TRAF6 complex

can lead to the activation of phosphatidylinositol 3-kinase (PI3K)/

protein kinase B (AKT) signaling pathway by recruiting Src

family kinases.(2,11,15,16) Activation of PI3K finally releases

intracellular Ca2þ,(17–19) which can activate the central transcrip-

tional factor in osteoclastogenesis, nuclear factor of activated

T-cells cytoplasmic 1 (NFATc1).(20) NFATc1 then translocates to

the nucleus and activates the expression of multiple osteoclas-

togenesis-related genes, such as tartrate-resistant acid phospha-

tase (TRAP), latent transforming growth factor beta binding

protein3 (LTBP3), chloride channel (ClC7), MMP9, calcitonin

receptor (CTR), cathepsin K, and c-Src.(21)

Caffeic acid 3,4-dihydroxy-phenethyl ester (CADPE), a com-

pound originally isolated frommedicinal plants Sarcandra glabra

and Teucrium pilosum,(22) has attracted much interest due to its

proven pharmacologic safety and its many biologic activities,

such as induction of cancer senescence,(23) inhibition of

tumor angiogenesis,(24) suppression of hepatocellular carcinoma

growth,(25) and gastric carcinoma cell migration.(26) In the

present study, we investigated the effects of CADPE on

osteoclastogenesis both in vitro and in vivo, and elucidated

the underlying molecular mechanisms. We found that CADPE

suppressed RANKL-induced osteoclast differentiation within

non–growth inhibitory concentrations at an early stage of

osteoclastogenesis. Addition of CADPE disrupted the formation

of the actin-ring during osteoclastogenesis in vitro, and

prevented ovariectomy-induced bone loss in vivo. Furthermore,

we found that CADPE inhibited NFATc1 expression by blocking

the MAPK/AP-1 signaling pathway and Ca2þ oscillation, but

not the NF-kB signaling pathway in RANKL-induced osteoclast

differentiation. Finally, CADPE decreased the expression levels

of osteoclast-related marker genes, including TRAP, cathepsin K,

and c-Src. Therefore, our data show that CADPE suppresses

osteoclastogenesis and osteoclast activity through MAPK/AP-1

and Ca2þ/NFAT signaling pathways.

Materials and Methods

Reagents and antibodies

CADPE was synthesized by the Department of Chemistry at

East China Normal University as described.(5) RAW264.7 cells

were a kind gift from Dr. B.G. Darnay (the University of Texas M.D.

Anderson Cancer Center, Houston, TX, USA). Penicillin, strepto-

mycin, and amodified essential medium (a-MEM) were obtained

from Invitrogen (Carlsbad, CA, USA), and fetal bovine serum (FBS)

was obtained from the FRONT Company. All antibodies were

purchased from Cell Signaling Technology (Beverly, MA, USA)

except the NFATc1 antibody, which was from Santa Cruz

Biotechnology (Santa Cruz, CA, USA). Fugene HD transfection

reagent (Roche, Shanghai, China) was used to transfect

RAW264.7 cells. TRIZOL reagent was purchased from Invitrogen

(Carlsbad, CA, USA), and the PrimeScript RT reagent Kit

was obtained from TaKaRa Biotechnology. Bacteria-derived

recombinant mouse RANKL and M-CSF were from R&D Systems

(Minneapolis, MN, USA). Tris, glycine, NaCl, SDS, and other

regents were obtained from Sigma (St. Louis, MO, USA).

Cytotoxicity assay

We detected the cytotoxic effect of CADPE by the sulforhoda-

mine B (SRB) assay method(27) using a VERSA max microplate

reader (Molecular Devices, Sunnyvale, CA, USA).

In vitro osteoclastogenesis assay

For primary cell cultures, we cultured bone marrow cells isolated

from C57/BL6 mice as described.(28) Briefly, BMMs were isolated

from femurs and tibias of 4-week-old C57BL/6 mice and cultured

in the presence of M-CSF (20 ng/mL) for 3 days. For differentia-

tion into mature osteoclasts, osteoclast precursors were cultured

with M-CSF (20 ng/mL) and RANKL (30 ng/mL) in 48-well culture

plates for 7 days with media changed every 2 days. For TRAP

staining, the cells were fixed and stained for TRAP activity

(Sigma). TRAP-positive multinucleated cells with >5 nuclei

were counted as osteoclasts. For RAW264.7 cells, culture was

performed as described.(28)

Actin-ring formation and pit assay

After stimulation with M-CSF (20 ng/mL) and RANKL (30 ng/mL),

we fixed the cells with 4% paraformaldehyde for 10 minutes

and washed them three times with PBS. Then the cells were

incubated with fluorescein isothiocyanate (FITC)-phalloidin for

Journal of Bone and Mineral Research CADPE INHIBITS RANKL-INDUCED OSTEOCLASTOGENESIS 1299

1 hour followed with 4,6-diamidino-2-phenylindole (DAPI)

staining for 10 minutes. For pit assay, osteoclasts were cultured

on FBS-coated dentin slices as described.(29)

Ovariectomized mouse model and bonehistomorphometric analysis

We created an ovariectomized mouse model as described.(30)

Maintenance, use, and treatment of all animals in this study were

in accordance with accepted standards of the Ethics Committee

at East China Normal University. Briefly, 7 days after ovariectomy,

mice were divided into three groups of six mice each: sham-

operatedmice (SHAM), ovariectomizedmice treated with vehicle

(OVX) and OVX mice treated with CADPE. CADPE (10mg/kg) or

dimethylsulfoxide (DMSO) was injected (intraperitoneally [i.p.])

into the ovariectomized mice every 2 days. After 3 months, all

mice were euthanized with excess amounts of anesthetic. The

body weight of these mice was measured every 2 days. Sections

of lumbar vertebrae (L3) were obtained for histomorphometric

measurements using the OsteoMeasure Analysis System

(Osteometrics, Decatur, GA, USA) according to standard criteria.

For osteoclast TRAP staining, calvariae were isolated and fixed in

10% paraformaldehyde fixation buffer (PFA) and decalcification

performed with 10% EDTA for 2 weeks. The samples were

embedded in paraffin for TRAP staining (Sigma).

Real-time RT-PCR

For the real-time RT-PCR analysis, we extracted total RNA

from cells with TRIZOL (Invitrogen) as described.(31) NFATc1,

cathepsin K, and TRAP transcripts were quantified on a Mx 3005P

(Stratagene, Santa Clara, CA, USA) using SYBR green dye and

normalized with b-actin. The following primer sets were used:

mouse TRAP: forward, 50-GCTGGAAACCATGATCACCT-30; reverse,50-GAGTTGCCACACAGCATCAC-30; mouse Cathepsin K: forward,

50-CTTCCAATACGTGCAGCAGA-30; reverse, 50-TCTTCAGGGCTTT-CTCGTTC-30; mouse NFATc1: forward, 50-TGGAGAAGCAGAGC-ACAGAC-30; reverse, 50-GCGGAAAGGTGGTATCTCAA-30; and

mouse b-actin: forward, 50-GTACGCCAACACAGTGCTG-30;reverse, 50-CGTCATACTCCTGCTTGCTG-30.

Luciferase reporter gene assay

The effect of CADPE on the RANKL-induced AP-1-luciferase

reporter assay was determined as described.(32,33)

Intracellular calcium imaging

We cultured BMMs in 35-mm cell culture dishes with or without

CADPE for 60 hours in the presence of RANKL (30 ng/mL) and

M-CSF (20 ng/mL). After washing with PBS, cells were incubated

with 5mM Fura-2/AM at 378C, and then imaged at 340

and 380 nM excitation wavelengths to detect intracellular free

calcium (with an Olympus IX71 and LAMBDA DG-4) and recorded

by InVivo software; analysis was by Image-Pro Analyzer 6.2

software (Media Cybernetics, Bethesda, MD, USA).(19)

Statistical analysis

All experimental data are presented as the mean� SD, with

values frommore than three experiments. Statistical significance

was determined by the Student t test.

Results

CADPE inhibits osteoclastogenesis in mouse BMMs andRAW264.7 cells

To examine the effects of CADPE on osteoclastogenesis, we used

two standard in vitro osteoclast differentiation models. The first

consisted of BMMs with RANKL and M-CSF stimulation. The

second was a mouse osteoclast precursor cell line RAW264.7

with RANKL stimulation.(28) BMMs were incubated with various

concentrations of CADPE during differentiation for 7 days, and

RAW264.7 cells with CADPE for 3 to 5 days. As shown in Fig. 1A–D,

CADPE inhibited osteoclast formation in a dose-dependent

manner in both BMMs and RAW264.7 cells, suggesting that

administration of CADPE in the differentiation cell models

inhibited osteoclast differentiation; with an one-half maximal

inhibitory concentration (IC50) of 0.5 to 1mM in BMMs and

RAW264.7 cells.

CADPE inhibits RANKL-induced actin-ring formationin osteoclasts

To further explore the effect of CADPE on osteoclastogenesis, we

tested whether CADPE could affect the actin-ring formation,

which is a prerequisite for osteoclast bone resorption and is the

most obvious character of mature osteoclasts during osteoclas-

togenesis.(34) In the presence of RANKL stimulation, BMMs can

differentiate into mature osteoclasts and form obvious actin-ring

structures by FITC-phalloidin staining (Fig. 1E). However, the size

and the number of actin-ring structures were significantly

decreased when cells were treated with 2.5 to 5mM CADPE

(Fig. 1E), suggesting that CADPE suppressed the formation of the

actin-ring in osteoclasts.

To investigate whether the blockage of osteoclastogenesis

and actin-ring formation by CADPE was due to potential toxicity

of this compound, we further examined the cytotoxicity of

CADPE by SRB assay on both BMMs and RAW264.7 cells

(Supplemental Fig. S1A and S1B). Although cytotoxicity was

observed when higher doses of CADPE were used, CADPE at

5mM (which completely inhibits osteoclastogenesis), did not

induce cytotoxicity significantly in both BMMs and RAW264.7

cells. These data indicate that the inhibitory effect of CADPE

on osteoclast differentiation was not due to the cytotoxicity of

the compound.

CADPE has no effect on M-CSF-induced proliferationand differentiation of BMMs

Osteoclastogenesis is a multistep process that can be separated

into two major events: the proliferation of BMMs and their

differentiation into osteoclast precursors induced by M-CSF;

and the subsequent differentiation of osteoclast precursors into

mature osteoclasts induced by RANKL.(12) To examine which step

CADPE affects in osteoclastogenesis, we first investigated the

effect of CADPE on BMM proliferation induced by M-CSF. Similar

to the cytotoxicity assay results, CADPE had little effect on

M-CSF-induced proliferation of BMMs at low concentrations (less

than 10mM) (Fig. 2A). To examine the effects of CADPE on BMM

differentiation into osteoclast precursors induced by M-CSF, we

investigated the expression levels of M-CSF–induced osteoclast

1300 WU ET AL. Journal of Bone and Mineral Research

precursor marker genes, RANK and c-Fms, using RT-PCR analysis.

As shown in Supplemental Figure S2, CADPE had little effect on

the expression of RANK and c-Fms induced by M-CSF. To confirm

this observation, we further examined the effects of CADPE

on cell surface expression of RANK by immunofluorescence

staining. Our results showed that CADPE had little effect on cell

surface expression of RANK as determined by the immunofluo-

rescence intensity or by the numbers of positively stained cells

(Fig. 2B). Together, these data suggested that the inhibitory

effect of CADPE on osteoclastogenesis was not due to

the suppression of M-CSF-induced BMM proliferation and

differentiation.

Fig. 1. CADPE inhibits osteoclastogenesis in BMMs and RAW264.7 cells.

(A, B) CADPE inhibits RANKL-induced mouse BMM differentiation. Mouse

BMMs (5� 103 cells) were incubated with M-CSF (10 ng/mL) and RANKL

(30 ng/mL), followed by treatment with or without the indicated doses of

CADPE. Cells were cultured for 7 days after RANKL stimulation and fixed

for TRAP assay. The cells were photographed (original magnification,

�100; left) and the numbers of TRAP-positive multinucleated (>5 nuclei)

osteoclasts were counted (right). Column, means of three experiments

performed in triplicate; bar, SD. (C, D) CADPE inhibits RANKL-induced

RAW264.7 cell differentiation. RAW264.7 cells (3� 103 cells) were incu-

bated with RANKL (30 ng/mL) followed by the addition of the indicated

concentrations of CADPE for 3 days and stained for TRAP expression.

The cells were photographed (original magnification, �100; left) and

the numbers of TRAP-positive multinucleated (>3 nuclei) osteoclasts

were counted (right). Column, means of three experiments performed in

triplicate; bar, SD. (E, F) CADPE suppresses RANKL-induced actin-ring

formation in mouse BMMs. BMMs (5� 103 cells) were incubated with or

without RANKL (30 ng/mL) in the presence of M-CSF (10 ng/mL), and cells

were then treated with or without CADPE (5mM) for 7 days. In E, Cells

were fixed and stained for F-actin. In F, Osteoclasts with actin-rings were

counted. Column, means of three experiments performed in triplicate;

bar, SD. bp< 0.01, cp< 0.001.

Fig. 2. CADPE has no effect on M-CSF-induced BMM proliferation and

differentiation, but inhibits RANKL-induced osteoclast formation at the

early stage. (A) BMMs (5� 103 cells) with M-CSF (10 ng/mL) cultured on

96-well plates and treated with various concentrations of CADPE for

3 days. Cell proliferation was measured after incubated with SRB as

described in Materials and Methods. Points, means of three experiments

performed in triplicate; bar, SD. (B) CADPE has little effect on M-CSF-

induced cell surface expression of RANK. BMMs were pretreated with or

without CADPE for 12 hours, and then stimulated with M-CSF (10 ng/mL)

for 48 hours. Cells were fixed and stained for RANK (left). Cells with

surface expression of RANK were counted (right). Column, means of

three experiments performed in triplicate; bar, SD. N.S.¼not significant.

(C, D) Effects of CADPE on RANKL-induced primary osteoclast precursor

differentiation at different time points (days). Osteoclast precursors

were cultured with RANKL (30 ng/mL) for differentiation into mature

osteoclasts. CADPE (5mM) was added at the indicated time (day). TRAP

staining was performed after RANKL stimulation for 7 days. The cells

were imaged (original magnification, �100 in C) and the numbers of

TRAP-positive multinucleated (>5 nuclei) osteoclasts were counted in D.

Column, means of three experiments performed in triplicate; bar, SD.

(E, F) Effect of CADPE on RANKL-induced RAW264.7 cell differentiation

at different time points. RAW264.7 cells (3� 103) were incubated with

RANKL (30 ng/mL), and then treated with CADPE (1mM) at the indicated

time (days). TRAP staining was performed after cell fixation. The cells

were imaged at different time points (original magnification, �100 in E),

and the numbers of TRAP-positive multinucleated (>3 nuclei) osteoclasts

were counted in F. Column, means of three experiments performed

in triplicate; bar, SD.


CADPE inhibits RANKL-induced osteoclast formation atthe early stage, but has little effect on mature osteoclastformation and resorption

To determine the effects of CADPE on RANKL-induced osteoclast

precursor differentiation into mature osteoclasts, we examined

the differentiation process of primary osteoclast precursors

beginning on day 0 to day 5 with RANKL stimulation in the

absence or presence of CADPE (Fig. 2C). Our results showed that

administration of CADPE strongly inhibited RANKL-induced

osteoclast differentiation during the first day of differentiation

(Fig. 2C, 1 day). However, CADPE was not effective in the

inhibition of osteoclast differentiation at later stages, especially

at day 5 (Fig. 2D). Similar results were obtained in RANKL-induced

RAW264.7 cell differentiation (Fig. 2E, F), suggesting that CADPE

inhibited RANKL-induced osteoclast differentiation at an early

stage.

To further explore whether CADPE inhibited osteoclast bone

resorption, we performed bone resorption assays using BMMs.

When BMMs were cultured on a dentin slice, mature osteoclasts

caused the resorption of lacunae and the formation of pits in the

present of M-SCF and RANKL, as compared to blank slice controls

(Fig. 3A, top left and middle). The number and area of pits on the

surface of the dentin slices were markedly decreased by the

addition of CADPE at the beginning of the assays (Fig. 3A, top

right). Similar results were obtained by confocal microscopy

scanning (Fig. 3A, bottom). These data were consistent with

previous results that CADPE inhibited osteoclast differentiation

and actin-ring formation, and as a result, CADPE inhibited

osteoclast activity. However, after RANKL stimulation for 6 days,

matured osteoclasts were formed and then seeded on dentin

slices, followed by incubation with or without CADPE for an

additional 2 days. The bone resorption lacunae pits and erosion

areas showed no significant differences between the control

slices and CADPE-treated slices (Fig. 3B), suggesting that CADPE

had little effect on mature osteoclast bone resorption. To further

investigate the effect of CADPE on mature osteoclasts, we

examined the sealing zone formation, which is essential to

osteoclast attachment to the bone and its resorption.(35) Our data

showed that CADPE exerted little effect on the sealing zone

dimensions, including the sealing zone width, perimeter, and

height (Supplemental Fig. S3C). Together, our data suggested

that CADPE exerted no effect on M-CSF-induced BMM

proliferation and differentiation, but inhibited RANKL-induced

osteoclast formation at the early stages.

CADPE prevents ovariectomy-induced bone loss byinhibiting osteoclast activity and stimulating osteoblastdifferentiation in vivo

Activation of osteoclasts plays an essential role in bone-related

diseases, including postmenopausal osteoporosis, in which

estrogen deficiency enhances the genesis and activity of

osteoclasts; and results in an unbalanced increase in bone

resorption.(36) To examine whether CADPE inhibits osteoclasto-

genesis in vivo, we used the ovariectomized mouse model

to mimic menopause-induced bone loss in women.(36) The

uterine weight data showed that the ovariectomy operation

was successful (Supplemental Fig. 4). Von Kossa staining and

histomorphometric analysis of lumbar vertebrae also indicated

that bone volume (BV/TV) and trabecular number (Tb.N) in the

ovariectomized (OVX) mice decreased dramatically, whereas

trabecular spacing (Tb.Sp) was dramatically increased when

compared with sham-operated controls (SHAM). Treatment of

CADPE (10mg/kg) in ovariectomized mice (OVXþCADPE)

significantly inhibited the ovariectomy-induced bone loss

as measured in these parameters (Fig. 4A, B). To investigate

whether CADPE prevented bone loss through inhibition of

Fig. 3. CADPE inhibits osteoclast bone resorption at early stages, but has

little effect on mature osteoclast bone resorption. (A) CADPE suppresses

osteoclast bone resorption at the early stage. BMMs (5� 103 cells) were

induced by M-CSF (10 ng/mL) and RANKL (30 ng/mL) with or without

CADPE (5mM) on dentine slices for 7 days. Dentine slices were stained

with Mayer’s hematoxylin after removal of cells. The resorption pits were

visualized with light microscopy (original magnification, �10; top) and

confocal microscopy (original magnification, �40; bottom). (B) CADPE

has little effect on bone resorption by matured osteoclasts. BMMs

(5� 103 cells) were induced by M-CSF (10 ng/mL) and RANKL (30 ng/

mL) for 6 days. Mature osteoclast cells were digested with collagenase

and seeded on dentine slices followed by treatment with or without

CADPE (5mM) for an additional 2 days. Dentine slices were stained with

Mayer’s hematoxylin after removal of cells. The resorption pits were

visualized with light microscopy (original magnification, �10) and con-

focal microscopy (original magnification, �40). The numbers of pits and

erosion areas were analyzed with Image-Pro Plus software (bottom).

Column, means of three experiments performed in triplicate; bar, SD.

N.S.¼not significant.


osteoclastogenic activity in vivo, we performed TRAP staining on

the long bone and calvarial bones of each group. The staining

data showed that the activity and area of osteoclasts in OVXmice

notably increased compared with sham controls, suggesting that

ovariectomy induced osteoclastogenesis and the activation of

osteoclasts. Treatment of OVX mice by CADPE dramatically

decreased the OVX-induced osteoclast activity (OVXþ CADPE

versus OVX mice) (Fig. 4C and Supplemental Fig. S5A).

Histomorphometric analysis confirmed that osteoclast surface/

bone surface (Oc.S/BS), eroded surface/bone surface (ES/BS), and

osteoclast number/bone perimeter (N.Oc/B.Pm) in OVX mice

increased markedly compared with sham controls (Fig. 4D).

These osteoclastic parameters were significantly decreased in

OVX mice treated with CADPE, as compared with the OVX mice

(Fig. 4D). Osteoclast activity was also confirmed by examination

Fig. 4. CADPE prevents ovariectomy-induced bone loss by inhibiting

osteoclast activity in vivo. (A) Representative von Kossa–stained sections

of lumbar vertebrae from sham, OVX, and OVXþ CADPE mice. (B) Bone

value/total value (BV/TV), trabecular space (Tb.Sp), and trabecular num-

ber (Tb.N) were analyzed with the OsteoMeasure Analysis System as

described in Materials and Methods (ap< 0.05, bp< 0.01, cp< 0.001

versus OVX group, n¼ 6). (C) Representative TRAP-stained histologic

sections of long bone from sham, OVX mice, and OVXþCADPE mice.

(D) Osteoclast surface/bone surface (Oc.S/BS), the number of osteoclasts/

bone perimeter (N.Oc/B.Pm), and the eroded surface/bone surface

(ES/BS) were analyzed with the OsteoMeasure Analysis System as de-

scribed in Materials and Methods. (E) CADPE enhances osteoblast differ-

entiation in vitro. BMSCs were isolated from female C57/BL6 mice and

treated with or without various doses of CADPE in the presence of

glycerol-phosphate, vitamin C, and dexamethasone for BMSC cell differ-

entiation into osteoblasts. The cells were stained with ALP solution at

day 4 and photographed. (F) CADPE has little effect on mouse body

weight at the concentrations tested. Body weight of different mouse

groups, including sham, OVX, and OVXþCADPE mice, were recorded

every 10 days (n¼ 6).

Fig. 5. CADPE suppresses the RANKL-induced MAPK/AP-1 signaling

pathway. (A) CADPE inhibits the phosphorylation of ERK, p38, and JNK

stimulated by RANKL. RAW264.7 cells were treated with or without

CADPE (5mM) for 4 hours, and then incubated with RANKL (100ng/mL)

for the indicated times. The cell lysates were extracted and subjected

to Western blot analysis with the indicated antibodies. (B) CADPE

suppresses RANKL-induced c-Fos protein nuclear translocation, but

has no effect on p65 protein nuclear translocation. RAW264.7 cells

were pretreated with the indicated concentrations of CADPE for 4 hours,

and then stimulated with RANKL for 20minutes. Cell nuclear extracts (NE)

and cytosolic extracts (CE) were collected and subjected to Western blot

analysis with the indicated antibodies. (C) CADPE inhibits the activity

of AP-1 induced by RANKL. RAW264.7 cells were transfected with the

AP-1-luciferase reporter gene and stimulated with RANKL for 16 hours

after pretreatment with the indicated concentrations of CADPE for

2 hours. Luciferase activity was measured as described in Materials

and Methods. Results are expressed as fold activity over the activity of

the control. Columns,means of three experiments performed in triplicate;

bars, SDs. (D) c-Fos prevents the inhibitory effect of CADPE in RANKL-

induced osteoclast differentiation. After transfection with c-Fos gene or

vector control plasmids, RAW 264.7 cells were treated with or without

CADPE (2.5mM) in the presence of RANKL. After 3 days, cells were fixed

and stained for TRAP (left). TRAP-positive multinucleated (>3 nuclei)

osteoclasts were counted (right). Columns, means of three experiments

performed in triplicate; bars, SDs; bp< 0.01.


for serum TRAP5b and cross-linked C-telopeptide 1 (CTX-1),

which reflects osteoclast activity in vivo. As shown in

Supplemental Fig. S5B, the serum TRAP5b and CTX-1 levels

were increased in OVX mice compared with sham-operated

control mice, whereas CADPE treatment significantly decreased

the levels of TRAP5b and CTX-1 induced by OVX, suggesting

that CADPE could inhibit ovariectomy-induced osteoclast activity

in vivo.

Because bone remodeling is regulated by both osteoblasts

and osteoclasts, we also investigated the effect of CADPE on

osteogenesis. CADPE strikingly enhanced osteogenic differenti-

ation of bone marrow stromal cells (BMSCs) in a concentration-

dependent manner as evaluated by alkaline phosphatase (ALP)

staining (Fig. 4E and Supplemental Fig. S5C). To comprehensively

evaluate the effect of CADPE on osteogenesis, in vivo ALP

staining was performed. As shown in Supplemental Fig. S5D,

CADPE markedly augmented osteoblast activity in OVX mice,

which is consistent with the in vitro osteogenic differentiation

assay.

Furthermore, to examine whether CADPE had a specific effect

on ovariectomy-induced bone loss, the effect of CADPE on sham-

operated mice was explored. Our data indicated that CADPE had

no significant effect on the bone parameters including BV/TV,

Tb.N, and Tb.Sp between the sham control mice and the CADPE-

treated sham mice (Supplemental Fig. S6), Moreover, in the

CADPE-treated groups, CADPE had little effect on body weight

at the concentrations tested at 3 months (Fig. 4F), which is

consistent with a previous report,(24) suggesting little toxicity of

CADPE at the tested concentration.

CADPE inhibits RANKL-mediated MAPK/AP-1signaling pathway

RANKL-induced MAPK/AP-1 activation is among the very early

molecular events induced by RANKL,(2,37) and our observations

showed that CADPE inhibited RANKL-induced osteoclast forma-

tion at an early stage of differentiation. To understand the

signaling pathways mediated by CADPE in osteoclastogenesis,

we examined whether CADPE suppressed RANKL-induced

phosphorylation of extracellular signal-regulated kinase (ERK),

p38, and c-Jun N-terminal kinase (JNK) by Western blot analysis.

The phosphorylation of MAPKs reached its peak within 30min

of RANKL stimulation (Fig. 5A, left). Treatment with CADPE

significantly decreased the phosphorylation of ERK, p38, and JNK

when compared with the RANKL-stimulated control (Fig. 5A). To

further investigate whether the formation of transcription factor

AP-1 was affected by CADPE, we examined the c-Fos nuclear

translocation in RAW264.7 cells with CADPE treatment. Our data

showed that nuclear translocation of c-Fos was upregulated with

RANKL stimulation, whereas the addition of CADPE inhibited the

nuclear translocation in a dose-dependent manner (Fig. 5B). To

confirm the data generated by Western blot assays, we also

performed an AP-1-luciferase reporter gene assay. CADPE

inhibited RANKL-induced AP-1 activity in a concentration-

dependent manner (Fig. 5C). To further confirm that CADPE

inhibited osteoclast differentiation by suppressing RANKL-

induced AP-1 activity, we investigated whether overexpression

of c-Fos in RAW264.7 cells could prevent the inhibition of CADPE

on osteoclast differentiation. CADPE inhibited osteoclast differ-

entiation in control RAW264.7 cells (Fig. 5D, top, and

supplemental Fig. S7A). However, CADPE had little inhibitory

effect on osteoclastogenesis in c-Fos-overexpressed RAW264.7

cells (Fig. 5D, bottom). To further examine whether CADPE

selectively targets the MAPK signaling pathway in osteoclasts,

we investigated the activation of MAPKs in mouse adipocyte

3T3-L1 cells with CADPE treatment using the same conditions.

Our data showed that CADPE had little effect on MAPK activation

in 3T3-L1 cells (Supplemental Fig. S7B), suggesting that CADPE

may specifically target the MAPK signaling pathway in

osteoclasts.

CADPE has little effect on RANKL-induced NF-kBsignaling pathway

RANKL-induced NF-kB signaling pathway is another pivotal early

molecular event induced by RANKL.(38) To determine whether

CADPE affects NF-kB activation, we investigated NF-kB activation

by Western blot assay using specific antibodies. Interestingly,

RANKL-induced phosphorylation of p65 and degradation of IkBa

were not affected with CADPE treatment (Fig. 6A). Furthermore,

nuclear translocation and DNA-binding activity of p65 were

not affected by CADPE treatment in nuclear translocation assay

(Fig. 5B) and electrophoretic mobility gel shift assay (EMSA)

(Supplemental Fig. S8A), suggesting that CADPE has little effect

on the RANKL-induced NF-kB signaling pathway.

CADPE suppresses RANKL-induced Ca2þ oscillation

Besides the MAPK/AP-1 and NF-kB pathways, RANKL-induced

Ca2þ oscillations constitute another early molecular event during

osteoclast differentiation.(11) To further understand the mecha-

nism of CADPE’s inhibitory effects on osteoclast differentiation,

cytosolic Ca2þ signals were investigated in BMMs. Our results

showed that RANKL markedly induced Ca2þ oscillations (Fig. 6B,

middle). However, addition of CADPE significantly inhibited

the amplitude and frequency of Ca2þ oscillations, and kept

the oscillations at a relatively low level (Fig. 6B, right, and

Fig. 6C).

CADPE inhibits RANKL-induced TRAF6-c-Src associationand AKT phosphorylation

Because release of intracellular Ca2þ requires activation of the

PI3K/AKT signaling pathway,(17–19) and RANKL activates PI3K/AKT

through the TRAF6 and c-Src complex.(15,16) we then assessed the

effect of CADPE on the RANKL-induced association of TRAF6 with

c-Src by endogenous coimmunoprecipitation assay and the

effect of CADPE on the RANKL-induced phosphorylation of

AKT by Western blot analysis. Our data showed that CADPE

suppressed the RANKL-induced interaction between TRAF6 and

c-Src in a dose-dependent manner (Supplemental Fig. S9A).

Similarly, CADPE dose-dependently inhibited RANKL-induced

AKT phosphorylation (Supplemental Fig. S9B). All of our results

suggest that CADPE suppresses RANKL-induced Ca2þ oscillation

by inhibition of the TRAF6/c-Src/PI3K/AKT pathway.


CADPE suppresses marker gene expression inosteoclastogenesis

RANKL has been shown to induce the expression of several

osteoclastogenesis-related marker genes, including NFATc1,

TRAP, cathepsin K, and c-Src. All of these are downstream genes

of NFATc1 that are regulated by MAPK/AP-1 and Ca2þ signaling

pathways. Because CADPE inhibited the RANKL-induced MAPK/

AP-1 pathway and Ca2þ oscillations, we next examined whether

CADPE inhibited expression of these osteoclastogenesis-related

genes. Our data showed that CADPE suppressed RANKL-induced

mRNA levels of NFATc1, TRAP, and cathepsin K in a concentration-

dependent manner (Fig. 7A). Furthermore, CADPE inhibited the

protein levels of NFATc1 and c-Src (Fig. 7B), suggesting that

CADPE inhibits marker gene expression in osteoclastogenesis,

which is regulated by the MAPK/AP-1 signaling pathway

and Ca2þ oscillations.

Discussion

Excessive RANKL signaling leads to enhanced osteoclast

formation and bone resorption, as seen in the pathologic bone

destruction of autoimmune arthritis, periodontitis, postmeno-

pausal osteoporosis, Paget’s disease, and bone tumors. There-

fore, downregulation of RANKL downstream signals is a proven

therapeutic method to the treatment of bone loss–related

diseases.(39) CADPE has been demonstrated to be effective in

inducing tumor cell senescence and suppression of tumor

growth, invasion, and angiogenesis.(23,24) However, its inhibitory

effect on osteoclastogenesis and the molecular mechanism(s)

are unknown. In this report, we demonstrated that CADPE

Fig. 6. CADPE has no affect on RANKL-induced NF-kB signaling pathway,

but inhibits RANKL-induced Ca2þ oscillations. (A) CADPE has little

effect on RANKL-induced p65 phosphorylation and IkBa degradation.

RAW264.7 cells were cultured in the presence of CADPE (5mM) for

4 hours, and then RANKL (100 ng/mL) was added at the indicated

time points. Cell lysates were extracted for Western blot analysis with

the indicated antibodies. (B, C) CADPE inhibits Ca2þ oscillation induced by

RANKL. BMMs were pretreated with or without CADPE (5mM), and then

incubated with or without RANKL for 60 hours. After loading with Fura-2

(2mM) for an additional 30 minutes, the cells were imaged at 340 and

380 nM excitation wavelengths to detect intracellular free calcium.

Ca2þ oscillations were recorded and analyzed with InVivo software as

described in Materials and Methods.

Fig. 7. CADPE represses the expression of RANKL-induced genes/pro-

teins involved in osteoclastogenesis. (A) CADPE inhibits the mRNA levels

of NFATc1, cathepsin K, and TRAP induced by RANKL. Total RNA was

isolated and analyzed by real-time PCR. Column, means of three experi-

ments performed in triplicate; bar, SD. (B) CADPE inhibits the protein

expression levels of NFATc1 and c-Src. BMMs were treated with or

without RANKL (30 ng/mL) plus various concentrations of CADPE for

3 days. Cell lysates were analyzed by Western blot with specific anti-

bodies. (C) The schematic model of the hypothesized mechanism by

which CADPE inhibits osteoclastogenesis and function. Activation of

RANK induces NFATc1 gene expression and activity through MAPK/

AP-1 and Ca2þ signaling pathways, and then regulates osteoclasto-

genesis related–gene expression, such as TRAP, NFATc1, c-Src, and

Cathepsin K. CADPE blocks RANKL-induced association of TRAF6-c-Src,

and then the activation of MAPK/AP-1 and Ca2þ/NFATc1 signaling, and

subsequently suppresses osteoclastogenesis related–gene expression.


inhibited RANKL-induced osteoclast formation and actin-ring

at the early stages but had no effect on M-CSF-induced BMM

proliferation and differentiation. CADPE also prevented ovariec-

tomy-induced bone loss through the suppression of osteoclast

activity in a mouse model. At the molecular level, CADPE

suppressed RANKL-induced phosphorylation of MAPKs, includ-

ing ERK, p38, and JNK, and the subsequent nuclear translocation

and activation of AP-1/c-Fos, as well as RANKL-induced Ca2þ

oscillations by inhibition of the TRAF6/c-Src/PI3K/AKT pathway;

however, CADPE had no effect on the NF-kB signaling pathway.

Finally, CADPE inhibited expression of osteoclastogenesis-

related marker genes, such as NFATc1, TRAP, cathepsin K, and

c-Src.

It is well established that M-CSF induces the proliferation of

BMMs and their differentiation into the osteoclast precursor,

whereas RANKL induces subsequent differentiation of osteoclast

precursors into mature osteoclasts. In our study, we demonstrat-

ed that CADPE had little effect on M-CSF–induced BMM

proliferation and osteoclast precursor marker gene expression,

whereas CADPE inhibited the RANKL-induced differentiation of

osteoclast precursors into osteoclasts. These data indicates that

CADPE suppressed RANKL-induced osteoclastogenesis at the

step of osteoclast precursor differentiation into mature osteo-

clasts, and suggests that CADPE modulated the RANKL signaling

pathway.

Binding of RANKL to its receptor RANK results in the

recruitment of the adaptor molecule’s tumor necrosis factor

receptor-associated factors (TRAFs), including TRAF1, 2, 3, 5,

and 6.(11) The NF-kB signaling pathway is one of the key

downstream signaling pathways from the complex.(1) Our

research indicates that CADPE had no effect on the degradation

of nuclear factor of kappa light polypeptide gene enhancer in

B-cells inhibitor, alpha (IkBa) and the phosphorylation, nuclear

translocation, and DNA-binding activity of NF-kB subunit p65. All

of the results suggest that CADPE had little effect on the RANKL-

induced NF-kB signaling pathway.

RANKL-induced intracellular Ca2þ oscillation requires the PI3K/

AKT signaling pathway(11,19,40) and RANKL activates PI3K/AKT

through the TRAF6 and c-Src complex in osteoclastogen-

esis.(15,16) In our study, we verified that CADPE inhibited the

RANKL-induced interaction between TRAF6 and c-Src, sup-

pressed the phosphorylation of AKT, and abrogated the

oscillation of Ca2þ, suggesting that CADPE modulated the

TRAF/c-Src/PI3K/Ca2þ signaling pathway in osteoclastogenesis.

Moreover, genetic and pharmacological evidence shows that

PI3K can regulate the activation of MAPKs induced by

RANKL.(16,41) In our results, CADPE suppressed the phosphoryla-

tion of MAPKs. Furthermore, CADPE inhibited AP-1/c-Fos nuclear

translocation and activity. When c-Fos was overexpressed in

RAW264.7 cells, the inhibitory effect of CADPE in osteoclasto-

genesis was partially prevented. These results suggested that

CADPE suppression of MAPKs signaling pathway was due to

CADPE regulation of the TRAF/c-Src/PI3K signaling pathway in

osteoclastogenesis.

Previous research showed that in ovariectomized mice,

a decrease in circulating estrogen levels led to production

of osteoclastogenic cytokines, such as TNFa and RANKL in

osteoblasts and T cells, in turn leading to rapid bone loss.(42)

Using an ovariectomized mouse model, we have demonstrated

through our histomorphometric analysis and TRAP staining

analysis that CADPE prevented bone loss by suppressing

osteoclastogenesis and osteoclast activity. We also examined

the effects of CADPE on bone homeostasis by investigating

osteoblast differentiation and osteoblast activity using OVX mice

and ALP staining, and demonstrated that CADPE increased

osteoblast differentiation. Therefore, CADPE prevented ovariec-

tomy-induced bone loss by inhibiting osteoclastogenesis and

increasing the formation of osteoblasts. The effects of CADPE in

bone homeostasis are similar to the widely used osteoporosis

drugs, bisphosphonates, which also inhibit bone digestion

by osteoclasts and stimulate the formation of osteoblast

precursors.(43) Therefore, further investigation will be needed

to understand the mechanism of CADPE’s involvement in

osteoblast differentiation.

Collectively, our data demonstrate for the first time, that

CADPE can suppress osteoclastogenesis both in vitro and in vivo.

Furthermore, we show that CADPE inhibits osteoclastogenesis

through inhibition of the MAPK/AP-1 signaling pathway

and Ca2þ oscillations, which lead to the activation of NFATc1

and the expression of downstream genes. These results suggest

that CADPE could be a potential therapeutic candidate in

treating osteoclast-related diseases such as osteoporosis.

Disclosures

All authors state that they have no conflicts of interest.

Acknowledgments

This work is partially supported by the grants from the National

Basic Research Program of China (2012CB910400, 2010CB529704),

National Natural Science Foundation of China (30930055,

30800653, 81071437, and 20802020), and the Science and Tech-

nology Commission of Shanghai Municipality (11DZ2260300).

Authors’ roles: Study design: XW, ML, and JL. Study conduct:

XW, ZL, ZY, CZ, and JJ. Data analysis: XW, ZL, ZY, YC, XY, XL, JX, ML,

and JL. Chemical compound synthesis: WQ, FY, and JT. Drafting

manuscript: XW. Revising manuscript content: ML, and JL. JL

takes responsibility for the integrity of the data analysis.

References

1. Boyle WJ, Simonet WS, Lacey DL. Osteoclast differentiation and

activation. Nature. 2003;423(6937):337–42.

2. Takayanagi H. Osteoimmunology: shared mechanisms and crosstalk

between the immune and bone systems. Nat Rev Immunol. 2007;7(4):292–304.

3. Teitelbaum SL, Ross FP. Genetic regulation of osteoclast develop-

ment and function. Nat Rev Genet. 2003;4(8):638–49.

4. Seeman E. Reduced bone formation and increased bone resorption:

rational targets for the treatment of osteoporosis. Osteoporos Int.

2003;14(Suppl 3):S2–8.

5. Zhang Z, Xiao B, Chen Q, Lian XY. Synthesis and biological evaluationof caffeic acid 3,4-dihydroxyphenethyl ester. J Nat Prod. 2010;73(2):

252–4.

6. Cappuzzo KA, Delafuente JC. Teriparatide for severe osteoporosis.

Ann Pharmacother. 2004;38(2):294–302.


7. Pazianas M, Abrahamsen B. Safety of bisphosphonates. Bone.2011;49(1):103–10.

8. Qiu SX, Dan C, Ding LS, Peng S, Chen SN, Farnsworth NR, Nolta J,

Gross ML, Zhou P. A triterpene glycoside from black cohosh that

inhibits osteoclastogenesis by modulating RANKL and TNFalphasignaling pathways. Chem Biol. 2007;14(7):860–9.

9. D’Amelio P, Grimaldi A, Di Bella S, Brianza SZ, Cristofaro MA, Tamone

C, Giribaldi G, Ulliers D, Pescarmona GP, Isaia G. Estrogen deficiencyincreases osteoclastogenesis up-regulating T cells activity: a key

mechanism in osteoporosis. Bone. 2008;43(1):92–100.

10. Cenci S, Toraldo G, Weitzmann MN, Roggia C, Gao Y, Qian WP,

Sierra O, Pacifici R. Estrogen deficiency induces bone loss by increas-ing T cell proliferation and lifespan through IFN-gamma-induced

class II transactivator. Proc Natl Acad Sci U S A. 2003;100(18):

10405–10.

11. Negishi-Koga T, Takayanagi H. Ca2þ-NFATc1 signaling is an essentialaxis of osteoclast differentiation. Immunol Rev. 2009;231(1):

241–56.

12. Takahashi N, Udagawa N, Tanaka S, Suda T. Generating murine

osteoclasts from bone marrow. Methods Mol Med. 2003;80:129–44.

13. Matsumoto M, Sudo T, Saito T, Osada H, Tsujimoto M. Involvement of

p38 mitogen-activated protein kinase signaling pathway in osteo-clastogenesis mediated by receptor activator of NF-kappa B ligand

(RANKL). J Biol Chem. 2000;275(40):31155–61.

14. Hotokezaka H, Sakai E, Kanaoka K, Saito K, Matsuo K, Kitaura H,

Yoshida N, Nakayama K. U0126 and PD98059, specific inhibitors ofMEK, accelerate differentiation of RAW264.7 cells into osteoclast-like

cells. J Biol Chem. 2002;277(49):47366–72.

15. Wong BR, Besser D, Kim N, Arron JR, Vologodskaia M, Hanafusa H,

Choi Y. TRANCE, a TNF family member, activates Akt/PKB througha signaling complex involving TRAF6 and c-Src. Mol Cell. 1999;4(6):

1041–9.

16. Lee SE, Woo KM, Kim SY, Kim HM, Kwack K, Lee ZH, Kim HH. Thephosphatidylinositol 3-kinase, p38, and extracellular signal-regulated

kinase pathways are involved in osteoclast differentiation. Bone.

2002;30(1):71–7.

17. Peng Q, Malhotra S, Torchia JA, Kerr WG, Coggeshall KM,Humphrey MB. TREM2- and DAP12-dependent activation of PI3K

requires DAP10 and is inhibited by SHIP1. Sci Signal. 2010;3(122):

ra38.

18. Boudot C, Saidak Z, Boulanouar AK, Petit L, Gouilleux F, Massy Z,Brazier M, Mentaverri R, Kamel S. Implication of the calcium sensing

receptor and the phosphoinositide 3-kinase/Akt pathway in the

extracellular calcium-mediated migration of RAW 264.7 osteoclast

precursor cells. Bone. 2010;46(5):1416–23.

19. Kuroda Y, Hisatsune C, Nakamura T, Matsuo K, Mikoshiba K.

Osteoblasts induce Ca2þ oscillation-independent NFATc1 activation

during osteoclastogenesis. Proc Natl Acad Sci U S A. 2008;105(25):8643–8.

20. Asagiri M, Sato K, Usami T, Ochi S, Nishina H, Yoshida H, Morita I,

Wagner EF, Mak TW, Serfling E, Takayanagi H. Autoamplification of

NFATc1 expression determines its essential role in bone homeostasis.J Exp Med. 2005;202(9):1261–9.

21. Takeshita S, Kaji K, Kudo A. Identification and characterization of the

new osteoclast progenitor with macrophage phenotypes being able

to differentiate into mature osteoclasts. J Bone Miner Res. 2000;15(8):1477–88.

22. El-Mousallamy AM, Hawas UW, Hussein SA. Teucrol, a decarboxyr-

osmarinic acid and its 40-O-triglycoside, teucroside from Teucrium

pilosum. Phytochemistry. 2000;55(8):927–31.

23. Dong A, Fang Y, Zhang L, Xie J, Wu X, Lian X, Chen Y, Luo J, Liu M.

Caffeic acid 3,4-dihydroxy-phenethyl ester induces cancer cell senes-

cence by suppressing twist expression. J Pharmacol Exp Ther. 2011;

339(1):238–47.

24. Jung JE, Kim HS, Lee CS, Park DH, Kim YN, Lee MJ, Lee JW, Park JW,Kim MS, Ye SK, Chung MH. Caffeic acid and its synthetic derivative

CADPE suppress tumor angiogenesis by blocking STAT3-mediated

VEGF expression in human renal carcinoma cells. Carcinogenesis.

2007;28(8):1780–7.

25. Won C, Lee CS, Lee JK, Kim TJ, Lee KH, Yang YM, Kim YN, Ye SK, Chung

MH. CADPE suppresses cyclin D1 expression in hepatocellular carci-

noma by blocking IL-6-induced STAT3 activation. Anticancer Res.2010;30(2):481–8.

26. Han H, Du B, Pan X, Liu J, Zhao Q, Lian X, QianM, LiuM. CADPE Inhibits

PMA-stimulated gastric carcinoma cell invasion and matrix metallo-

proteinase-9 expression by FAK/MEK/ERK-mediated AP-1 activation.Mol Cancer Res. 2010;8(11):1477–88.

27. Mesa-Siverio D, Machin RP, Estevez-Braun A, Ravelo AG, Lock O.

Structure and estrogenic activity of new lignans from Iryanthera

lancifolia. Bioorg Med Chem. 2008;16(6):3387–94.

28. Li C, Yang Z, Li Z, Ma Y, Zhang L, Zheng C, Qiu W, Wu X, Wang X, Li H,

Tang J, Qian M, Li D, Wang P, Luo J, Liu M. Maslinic acid suppresses

osteoclastogenesis and prevents ovariectomy-induced bone loss by

regulating RANKL-mediated NF-kappaB and MAPK signaling path-ways. J Bone Miner Res. 2011;26(3):644–56.

29. Koide M, Kinugawa S, Ninomiya T, Mizoguchi T, Yamashita T, Maeda

K, Yasuda H, Kobayashi Y, Nakamura H, Takahashi N, Udagawa N.Diphenylhydantoin inhibits osteoclast differentiation and function

through suppression of NFATc1 signaling. J Bone Miner Res. 2009;

24(8):1469–80.

30. Luo J, Zhou W, Zhou X, Li D, Weng J, Yi Z, Cho SG, Li C, Yi T, Wu X, LiXY, de Crombrugghe B, Hook M, Liu M. Regulation of bone formation

and remodeling by G-protein-coupled receptor 48. Development.

2009;136(16):2747–56.

31. Yang Z, Li C, Wang X, Zhai C, Yi Z, Wang L, Liu B, Du B, Wu H, Guo X, LiuM, Li D, Luo J. Dauricine induces apoptosis, inhibits proliferation and

invasion through inhibiting NF-kappaB signaling pathway in colon

cancer cells. J Cell Physiol. 2010;225(1):266–75.

32. Ikeda F, Nishimura R, Matsubara T, Tanaka S, Inoue J, Reddy SV, Hata

K, Yamashita K, Hiraga T, Watanabe T, Kukita T, Yoshioka K, Rao A,

Yoneda T. Critical roles of c-Jun signaling in regulation of NFAT

family and RANKL-regulated osteoclast differentiation. J Clin Invest.2004;114(4):475–84.

33. Li C, Yang Z, Zhai C, QiuW, Li D, Yi Z, Wang L, Tang J, QianM, Luo J, Liu

M. Maslinic acid potentiates the anti-tumor activity of tumor necrosis

factor alpha by inhibiting NF-kappaB signaling pathway. Mol Cancer.2010;9:73.

34. Wilson SR, Peters C, Saftig P, Bromme D. Cathepsin K activity-

dependent regulation of osteoclast actin ring formation and bone

resorption. J Biol Chem. 2009;284(4):2584–92.

35. McMichael BK, Meyer SM, Lee BS. c-Src-mediated phosphorylation of

thyroid hormone receptor-interacting protein 6 (TRIP6) promotes

osteoclast sealing zone formation. J Biol Chem. 2010;285(34):26641–51.

36. Sun L, Vukicevic S, Baliram R, Yang G, Sendak R, McPherson J, Zhu LL,

Iqbal J, Latif R, Natrajan A, Arabi A, Yamoah K, Moonga BS, Gabet Y,

Davies TF, Bab I, Abe E, Sampath K, Zaidi M. Intermittent recombinantTSH injections prevent ovariectomy-induced bone loss. Proc Natl

Acad Sci U S A. 2008;105(11):4289–94.

37. Rosenberger SF, Finch JS, Gupta A, Bowden GT. Extracellular signal-

regulated kinase 1/2-mediated phosphorylation of JunD and FosB isrequired for okadaic acid-induced activator protein 1 activation. J Biol

Chem. 1999;274(2):1124–30.

38. Iotsova V, Caamano J, Loy J, Yang Y, Lewin A, Bravo R. Osteopetrosis

in mice lacking NF-kappaB1 and NF-kappaB2. Nat Med. 1997;3(11):1285–9.

39. Leibbrandt A, Penninger JM. RANK/RANKL: regulators of immune

responses and bone physiology. Ann N Y Acad Sci. 2008;1143:

123–50.


40. Zou W, Kitaura H, Reeve J, Long F, Tybulewicz VL, Shattil SJ, GinsbergMH, Ross FP, Teitelbaum SL. Syk, c-Src, the alphavbeta3 integrin, and

ITAM immunoreceptors, in concert, regulate osteoclastic bone

resorption. J Cell Biol. 2007;176(6):877–88.

41. Munugalavadla V, Vemula S, Sims EC, Krishnan S, Chen S, Yan J, Li H,

Niziolek PJ, Takemoto C, Robling AG, Yang FC, Kapur R. The p85alphasubunit of class IA phosphatidylinositol 3-kinase regulates the

expression of multiple genes involved in osteoclast maturationand migration. Mol Cell Biol. 2008;28(23):7182–98.

42. Krum SA, Brown M. Unraveling estrogen action in osteoporosis. Cell

Cycle. 2008;7(10):1348–52.

43. Jin H, Dai J, Chen X, Liu J, Zhong D, Gu Y, Zheng J. Pulmonary toxicity

and metabolic activation of dauricine in CD-1 mice. J Pharmacol ExpTher. 2010;332(3):738–46.


Caffeic acid 3,4-dihydroxy-phenethyl ester suppresses receptor activator of NF-κB ligand-induced osteoclastogenesis and prevents ovariectomy-induced bone loss through inhibition of

Documents